BR112020021267A2 - particle sedimentation devices - Google Patents

Abstract

The present invention relates to sedimentation devices for separating particles from a bulky fluid with applications in numerous fields. The particle sedimentation devices include a first stack of cones with a small opening oriented upwards or downwards. Optionally, sedimentation devices may include a second stack of cones with a small opening facing downwards or upwards. Cones can be concave or convex. These devices are useful for separating (in sizes in millimeters or microns) small particles of a bulky fluid with applications in numerous fields, such as cultures of biological cells (microbials, mammals, plants, insects or algae), separation of solid catalyst particles treatment of a liquid or gas and waste water.

Description

Invention Patent Descriptive Report for "PARTICULATE SEDIMENTATION DEVICES".

CROSS REFERENCE AT RELATED PATENT APPLICATION

[0001] This patent application claims the priority benefit under 35 USC, 8119 (e) for Provisional Patent Application serial number US 62 / 659,295, filed on April 18, 2018, and is related to Patent Application No. US 15 / 586,902, filed on May 4, 2017, whose Application is a partial continuation of Patent Application No. US 15 / 324,062, filed on January 5, 2017, and for PCT Application No. PCT / US2015 /063,195, which has an international filing date of December 1, 2015, and which designated the United States. This Order is also related to Provisional Patent Application No. US 62 / 332,546, filed on May 6, 2016, and to Provisional Patent Application No. US 62 / 459,509, filed on February 15, 2017. The Patent Application US No. 15 / 324,062 is a National Internship Application under 35 USC 371 of PCT Application No. PCT / US2015 / 039,723, which has an international filing date of July 9, 2015, which designated the United States, whose PCT Application claims the benefit of Provisional Patent Application No. US 62 / 022,276, filed on July 9, 2014, and Provisional Patent Application No. 62 / 037,513, filed on August 14, 2014. PCT Application No. POT / US2015 /063,195 claims the benefit of Provisional Patent Application No. 62 / 086.122, filed on December 1, 2014. All of these Orders are incorporated in this document for reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention features cell or particle sedimentation devices with improved sedimentation on the inclined surfaces of multiple layers. The devices of the present invention have applications in numerous fields, including:

(i) cultures of biological cells with high cell density (mammals, microbials, plants or algae) that secrete polypeptides, hormones, proteins or glycoproteins, vaccines or particles similar to vaccines or other small chemicals, such as ethanol, isobutanol , isoprenoids, flavoring and fragrance compounds, etc .; (ii) separation and recycling of solid porous or non-porous catalyst particles that catalyze chemical reactions in the liquid or gas phase that surround the solid particles; (ili) separation and collection of solids recently formed in physical transformations, such as crystallization, flocculation, agglomeration, precipitation, etc., from the surrounding liquid phase; (iv) capture and purification of secreted proteins, such as monoclonal antibodies, and others, in affinity binders, such as protein A immobilized in microspherical granules; (v) in vitro expansion of various mammalian cells, such as human mesenchymal stem cells, differentiated human cells (eg, cardiomyocytes or red blood cells), modified human cells (eg, T lymphocytes transfected with antigen receptor) chimeric or CAR-T cells, etc., for autologous or allogeneic cell therapy applications); and (vi) water clearing processes in large municipal or commercial wastewater treatment plants by sedimentation and removal in a complex biological pool or activated sludge or other solid particles.

DESCRIPTION OF RELATED TECHNIQUE

[0003] Of all the fields of application of the aforementioned sedimentation devices, the most immediately applicable well-established field is the production of biological proteins, polypeptides or hormones secreted from cultures suspended in microbial cells or from recombinant mammals. The most common methods of producing biological proteins in microbial or recombinant mammalian cells are based on fed batch cultures, in which the cells are grown at high cell densities and then normally exposed to an induction medium or a inducer to provoke the production of proteins. If the desired proteins are secreted out of cells, it is more cost-effective to switch from a fed batch culture to a continuous perfusion culture that can maintain high cell density and high productivity over a much longer duration. of culture. During continuous perfusion cultures, live and productive cells are retained or recycled back into the bioreactor while secreted proteins are continually harvested from the bioreactor for downstream purification processes.

[0004] Some of the key advantages of cultures in continuous perfusion over fed batch cultures are: (1) secreted protein products are continuously removed from the bioreactor, without subjecting these products to potential degradation by enzymes proteolytics and / or glycolytics released in the culture medium of dead cells; (2) live, productive cells are retained or recycled again to achieve high cell densities in continuous perfusion bioreactors, where they continue to produce valuable proteins within the controlled environment of the bioreactor for a much longer culture life , instead of being exterminated and removed from the bioreactor at the end of each fed batch culture; (3) the environment of the perfusion bioreactor can be maintained much closer to the conditions of the constant state (thereby maintaining a more consistent product quality through design) with the continuous addition of fresh nutrient media and the removal of waste products together with the harvested protein products, unlike the concentrations of nutrients and waste products in dynamic change in the fed batch culture; and (4) with a subset of cell retention devices, the smallest dead or dying cells can be selectively removed from the perfusion bioreactor before these cells smooth and release their intracellular enzymes, thereby maintaining a high fraction of cell viability and a high quality of secreted protein products while being harvested.

[0005] Many cell retention devices have been developed in the mammalian cell culture industry, such as internal rotary filter devices (Himmelfarb et al., Science 164: 555-557, 1969), filtration modules external (Brennan et al., Bio-technol. Techniques, 1 (3): 169-174, 1987), hollow fiber modules (Knazek et al., Science, 178: 65-67, 1972), gravitational sedimentation - nal in a cyclone (Kitano et al., Appli. Microbiol Biotechnol. 24, 282- 286, 1986), inclined sedimenters (Batt et al., Biotechnology Progress, 6: 458-464, 1990), continuous centrifugation ( Johnson et al., Biotechnology Progress, 12, 855-864, 1999) and acoustic filtration (Gorenflo et al., Biotechnology Progress, 19, 30-36, 2003). Cyclones have been shown to be unable to produce sufficient centrifugal force to separate cells at device sizes and harvest rates used in mammalian cell culture experiments (Kitano et al., 1986) and mammalian cells are seriously damaged at higher flows (and centrifugal forces) needed for efficient cell separation (Elsayed, et al. Eng. Life Sci, 6: 347-354, 2006). While most other devices properly retain all mammalian cells from the harvest, these devices are unable to separate the dead cells from the desired living cells in the bioreactor. As a result, dead cells continue to accumulate inside the perfusion bioreactor and membrane filters become clogged, requiring the completion of the continuous perfusion bioreactor, usually within three or four weeks of mammalian cell culture.

[0006] Among all the cell retention devices available today, only the inclined sedimenters (Batt et al., 1990, supra, and Searles et al., Biotechnology Progress, 10: 198-206, 1994) per - mitigate the selective removal of smaller dead cells and cell residues in the upper flow or in the harvest flow, while the cells of large, living and productive mammals are continuously recycled through the lower flow again in the perfusion bioreactor. Therefore, it is feasible to continue operating the perfusion bioreactor indefinitely at high viability and high cell densities while the protein product is continuously harvested from the top of the inclined sedimentator.

[0007] The inclined settler was previously enlarged as a multi-plate or lamellar settler (Probstein, RF, US Patent No. 4,151,084, April 1979) and used extensively in several large-scale industrial processes, such as treatment - wastewater treatment, clearing drinking water, finishing metals, mining and catalyst recycling (eg Odu- eyngbo et al., US Patent No. 7,078,439, July 2006).

[0008] In reference to our first demonstration of a single inclined plate sedimentator (Batt et al., 1990) to improve the productivity of secreted proteins in mammalian cell culture applications, a multi-plate or lamellar sedimentation device has been patented in order to expand the inclined sedimenters for use in hybridoma cell culture (Thompson and Wilson, US Patent No. 5,817,505, October 1998). Such lamellar inclined sedimentation devices have been used to grow recombinant mammalian cells in continuous perfusion bioreactors with high productivity of the bioreactor (due to the high density of the cells) and high viability (> 90%) for a long time (for example , several months without any need to finish the culture in perfusion). U.S. Patent Application No. 2011/0097800, granted to Kauling et al., Describes an enlarged version of inclined sedimenters that use cylindrical tubes wrapped at inclined angles. The device is described as being useful in the cultivation of larger mammalian cells, such as CHO, BHK, HEK, HKB, hybridoma cells, ciliate and insect cells.

[0009] None of these cell retention devices have been demonstrated in the collection of protein products secreted in cultures of the perfusion bioreactor in smaller cells and, even more challenging, in microbial ones. Lamellar sedimenters have been tested with yeast cells to investigate cell sedimentation with limited success (Bungay and Millspaugh, Biotechnology and Bioengineering, 23: 640-641, 1984). Hydrocyclones have been tested in yeast suspensions, mainly to separate yeast cells from beer, and again only with limited success (Yuan et al., Bioseparation, 6: 159-163, 1996; Cilliers and Harrison, Chemical Engineering Journal, 65: 21-26, 1997).

[0010] A modified cyclone with a vertical spiral plate inside the cyclone has been proposed in order to improve the separation efficiency in wastewater treatment (Boldyrev VV, Davydov El, Settling tanks, as described in Russian Patent No. 2,182,508) , and an earlier description of this arrangement has been described for the decantation of solids in liquid suspension (US Patent No. 4,048,069, September 1977). The modified cyclone described in Russian Patent No. 2,182,508 includes a spiral-wound plate contained in a vertical cylindrical drum with a tapered bottom. A slit is provided along the entire height of a central waste water inlet pipe, which is plugged at the bottom in order to channel the waste water from the inlet pipe into the vertical spiral wound plate. The spiral starts at the

bo central and ends at the wall of the cylindrical casing, forming a channel through which particles-laden wastewater flows. The particles settle in the vertical settling column of the spiral channel. The height of the pelletizer zone is the vertical height of the spiral plate and the width of the channel is formed by the walls of the spiral-wound plate, which is kept constant throughout its length. A pipe to remove the purified water is installed in the upper part of the cylindrical body. A conduit to remove the sediment is installed at the bottom of the lower conical portion. In operation, the residual water enters through the central tube and enters the spiral zone through the crack or opening. The spiral channel serves to increase the flow path and thus to increase the residence time of the liquid in the sedimenter. The spiral also serves to increase the contact area (wall) for the fluid. The main driving force in lightening is gravity, which acts on the particles of the suspension as the suspension surrounds the vertical sedimentation column wound in a spiral. The paste that is left on the spiral wall or in the channel falls on the conical bottom of the sedimenter and is periodically removed from the sedimenter. The purified water is extracted through a pipe on the side of the cylindrical enclosure near the top.

[0011] As described in the Russian patent document, the flow pattern of wastewater containing solids is inverted from the typical flow pattern of a common cyclone, as dirty water enters the center through the central tube, and enters the spiral channel through the slits, and the purified water is removed from the periphery or outside of the vertical cylindrical body through a purified water pipe. This modified inverted flow cyclone device was neither proposed nor applied to any other fields, with the exception of waste water treatment.

[0012] In this way, a particle sedimentation device that can potentiate centrifugal forces and gravitational forces in particles in liquid suspension in a relatively small space is desired.

SUMMARY

[0013] The present invention features cell or particle sedimentation devices with improved sedimentation on inclined, multi-layered surfaces arranged within an enclosure. The wrapper can be a cyclone wrapper. The particle separation devices of the present invention can be used in various applications and represent a major improvement over the prior art separation devices. In these sedimentation devices, the inclined surfaces can be attached to a plurality of vertical cylindrical plates. The sedimentation devices may include a tapered spiral surface or several inclined plates that approach the angled tapered surface connected to the bottom of a spiral. The various angled, layered plates enhance the sedimentation efficiency of the bulky fluid particles that move downwards or upwards within a conical assembly in which the liquid volume moves progressively from the periphery of the conical or spiral sedimentation surfaces to the center of the sedimentation device.

[0014] The sedimentation devices of the present invention may include a casing that includes a series of stacked cones positioned inside the casing, tapering downwards towards a central opening, without any vertical plate. The cones of the present modality are supported on the stack, one on top of the other, by supports that maintain a distance (or channel width) between the successive cones in the stack. The supports can comprise three or more projections fixed to the upper and / or lower surface of one or more of the cones to position the successive cones at a desired distance

(the desired channel width). Optionally, the supports can comprise at least three L-shaped elements interconnected to a surface of each cone that is distal to the truncated apex of the cone. The L-shaped elements include a first side interconnected to a second side at an apex and are interconnected to the surface so that the first side supports a second cone on the cone cone. The second side is substantially parallel to the surface of the cone. Optionally, the second side can project beyond the cone to space the cone from an inner surface of the enclosure. In some embodiments, there are no plugs or other impediments that impede the flow of liquid or suspended particles from the stacked conical surfaces to the central opening.

[0015] The sedimentation devices of the present invention may include a housing that includes: 1) a first stack of two or more stacked cones, each of which has a central opening, and, 2) an optional second stack of two or more stacked cones, each of which has a central opening, joined at or near its bottom, with conical surfaces tapering downwards towards a central opening at the bottom of the housing.

[0016] The stacked cones (both in the first and the optional second stack of two or more stacked cones) comprise at least three projections that support each cone above the next successive cone in the stack. Preferably, the projections are placed at a substantially constant distance and formed in a generally equal size to maintain each successive cone in the pile with an equal spacing between all the cones in the pile. In a modality, there are at least three projections for each cone in order to correctly support each successive cone, but each cone can comprise more than three projections, as necessary to properly or correctly support the cone. For example, each cone can comprise four projections or it can comprise eight projections, to support the next successive cone in the stack.

[0017] The projections, or "vertical supports", can represent an impediment for the particles or sedimented cells to slide down from the surface of a cone to the central opening or to the space around the inner circumference of the envelope between the casing and cones. These projections are joined to a surface of a cone, but these projections do not need to join to another cone in the cone stack. Thus, these projections do not need, and in most modalities, they do not join two or more cones in a pile to each other.

[0018] Preferably, there is a substantially constant spacing between each successive conical surface created by the projections that support each successive cone in a pile of cones. The spacing between successive cones can vary from about 1 mm to about 2.5 cm.

[0019] This arrangement of sedimentation surfaces, provided by successive stacks of cones, in which each is supported by the next successive cone, but is not permanently attached to the next successive cone, is particularly useful for separation applications where the particle sedimentation device, and the conical surfaces therein, require regular or continuous service, such as disassembly and cleaning the conical sedimentation surfaces within the sedimentation device.

[0020] This arrangement of a first and an optional second stack of cones significantly improves the sedimentation efficiency of the particles of a bulky fluid as the bulky fluid moves through the sedimentation device. As the bulky liquid, which includes particles such as cells, moves through the stacked cones of the sedimentation device of the present invention, the larger particles (for example, live and productive cells) sediment on the surface of the cones. The cells that slide down from the top or the first stack of cones slide down from the conical surfaces to the outer edges of the cones and fall vertically into the conical section of the casing. In addition, the cells that slide down from the bottom stack or second stack of cones, slide down from the conical surfaces to the central opening of the cones and fall vertically into the central opening of the housing.

[0021] These devices can be enlarged or reduced to meet the needs of different industries or applications or sizes when the separation surface is enlarged or reduced volumetrically in three dimensions, compared to a more uni or bidimensional dimensioning typical of previous sedimentation devices.

[0022] The enlargement of the devices of the present invention can be carried out simply by increasing the diameter of the casing (and, correspondingly, by increasing the diameter of the cones stacked inside) and / or by increasing the height of the casing (which increases the number of cones in one or both of the first and second piles of cones). The effective projected area for cell sedimentation increases proportionally to the square of the envelope diameter and increases proportionally to the height of the inner cylinders. The effective settling area of the compact settling devices of the present invention increases proportionally to the cube of the casing diameter (assuming that the height of the inner settler is also proportionally increased) or equivalent to the casing volume. This three-dimensional or volumetric enlargement of an effective sedimentation area makes the sedimentation device of the present description much more compact compared to the previous inclined sedimentation devices.

[0023] The radial spacing in the annular regions between the different cylinders or cones can be between about 1 cm to about 10 cm, ideally around 2.5 cm. A small spacing of about 1 mm between the inclined sediment cones and the inner surface of the next successive cone provides a useful space for the sedimented particles (eg cells) to slide under the surface of the cones and out of the cones from the side, instead of sliding down to the bottom of the cone. The cells that come out from the sides settle vertically along the inside of each cylinder. When these sedimentation cells reach the tapered surface at the bottom of each cylinder, they slide downward, on the sloping surface in the cone, to the central opening at the bottom of the cyclone shell. An advantage of increasing fluid velocity as they descend from the inclined conical surface to the central opening is that an increasing number of sedimented cells that slide down the cone are dragged downward to the central opening, rather than being allowed to accumulate through the faster liquid speeds.

[0024] The angle of inclination for the sedimentation surfaces may or may not be constant, varying between about 15 degrees to about 75 degrees in relation to the vertical. For use with more sticky particles (usually mammalian cells), the angle of inclination can be closer to the vertical (ie, around 15 degrees from the vertical). For use with solid, non-sticky catalyst particles, the angle of inclination may be further away from the vertical (for example, around 75 degrees from the vertical). In some embodiments, the conical surfaces have an arched longitudinal cross-section so that the angle of inclination varies with respect to the longitudinal axis between about 10 degrees to about 80 degrees, or about 15 degrees to about 75 degrees.

[0025] All sedimentation devices of the present invention may include a closure or cover over at least a portion of the enclosure at an end of the enclosure opposite the first opening. In all of these embodiments, the closure or lid may also include an outlet or port for removing liquids or for entering liquids into the sedimentation device. The opening and additional ports or outlets in the housing and / or cover are in liquid communication with the outside and the inside of the housing to allow liquids to pass into and / or out of the sedimentation device housing , and in each instance of such an opening or inlet / outlet, these passageways into and out of the enclosure may include valves or other mechanisms that can be opened or closed to interrupt or restrict the flow of liquids in or out. outside the sedimentation devices of the present invention.

[0026] The particle sedimentation devices of the present invention can include an enclosure and at least one vertical tube disposed within the enclosure, and at least one vertical tube attached to one end with a tapered surface that tapers downward for a first opening. in the cyclone shell. There is at least one additional opening in the housing substantially opposite the first opening.

[0027] The angle of inclination for the conical surfaces is about 45 degrees in relation to the vertical in a modality or it can vary between about 15 degrees in relation to the vertical and about 75 degrees in relation to the vertical. Optionally, the conical surfaces and / or the upper or lower part of the enclosure can have a concave or convex shape, so that the angle of inclination varies between about 15 degrees in relation to the vertical and about 75 degrees in relation to the vertical.

[0028] The width of an annular ring channel formed between the adjacent vertical tubes is between about 1 mm and about 50 mm. The number of vertical tubes within the sedimentation device can be between about 2 and about 30.

[0029] The sedimentation device may include a closure over at least a portion of the housing at an end of the housing opposite the first opening. At least one additional opening in the enclosure can be configured to open from one side of the enclosure tangentially to at least one vertical tube, in liquid communication with the exterior and interior of the enclosure.

[0030] A liquid harvest outlet can be formed in the closure, in net communication with the outside and inside of the enclosure.

[0031] Another aspect of the present invention is a particle sedimentation device that can include, but is not limited to, a casing that includes one or more of: (1) a first conical portion; (2) a second conical portion; (3) a cylindrical portion located between the first and second conical portions; (4) at least one inlet to introduce liquid into the enclosure; (5) a first exit door; (6) a second exit port; and (7) a first stack of cones located within the casing. In one embodiment, the first exit port is associated with the first conical portion and the second exit port is associated with the second conical portion. Optionally, the liquid introduced into the enclosure can be a liquid suspension that includes particles. The particles can have a plurality of sizes.

[0032] In one embodiment, the first outlet port can be used to collect a clarified liquid. The clarified liquid can

include a first subset of particles. The first subset of particles can comprise cell residues, dead cells and others. Optionally, the first exit door can be formed by closing the enclosure. The first exit port is in liquid communication with the outside and inside of the enclosure.

[0033] Optionally, in another modality, the second outlet port can be used to collect a concentrated liquid. The concentrated liquid can include a second subset of particles, such as living cells. Usually, the particles in the second particle subset are generally larger than the particles in the first particle subset. Each particle of the second subset of particles generally has a greater mass than the particles of the first subset of particles. The second outlet port is in liquid communication with the outside and inside of the enclosure.

[0034] The first pile of cones occupies at least part of the first conical portion. Optionally, the first stack of cones occupies at least a portion of the cylindrical portion. Optionally, one or more cones from the first cone stack include a truncated apex oriented towards the first exit port. Additionally, or alternatively, at least one cone from the first pile of cones is devoid of the central opening. In another embodiment, each cone in the first stack of cones includes an open base oriented towards the second exit port. The cones of the first cone stack are generally centered in the casing, for example, the cones of the first cone stack can be centered around a substantially central opening formed by the truncated apex of one or more cones.

[0035] Optionally, the casing may also include a second stack of cones. The second stack of cones can occupy at least part of the second conical portion and can occupy at least part of the cylindrical portion. In one embodiment, each cone in the second

of the cone stack is transversal to the cones of the first cone stack.

[0036] Optionally, an angle of inclination to the surface of a cone in the first pile of cones can vary between about 15 degrees to about 75 degrees in relation to the vertical. In one embodiment, the surface of a cone is convex or concave, so that a cross section of the cone's surface defines an arcuate line. In another modality, the angle of inclination of the cones can be constant at any angle between 15 and 75 degrees in relation to the vertical. In one embodiment, the tilt angle of the cones is about 45 degrees.

[0037] In another modality, each cone of the second stack of cores includes a truncated apex oriented towards the second exit port. Each cone in the second stack of cones can also include an open base oriented towards the first outlet port. In one embodiment, the cones in the second stack of cones are usually centered in the envelope. In another embodiment, the cones of the second pile of cones are centered around a substantially central opening formed by the truncated apex of one or more cones.

[0038] In one embodiment, an angle of inclination to the surface of a cone in the second pile of cones is between about 15 degrees to about 75 degrees in relation to the vertical. The angle of inclination of the cones in the second stack of cones can be about 45 degrees.

[0039] In one embodiment, the cones of the first pile of cones have a substantially uniform spacing. In addition, the cones of the second cone stack can have substantially uniform spacing. In one embodiment, the cones of the first cone stack have a different spacing compared to the cones of the second cone stack.

[0040] At least one entry is configured as a gateway in liquid communication with the outside and inside of the enclosure. At least one entry can be associated with at least one of the first conical portion, the second conical portion and the cylindrical portion of the housing. In one embodiment, a first entry of at least one entry is associated with the cylindrical portion of the housing. In another modality, a second entry of at least one entry is associated with one of the first and second conical portions. In another modality, the second entry is associated with the second conical portion. In another mode, at least one input is configured to be interconnected to a disposable bag from the bioreactor. The disposable bioreactor bag can comprise a plastic material.

[0041] A configuration of the particle sedimentation devices of the present invention may include an enclosure, which comprises: (a) a first conical portion; (b) a second conical portion; (c) a cylindrical portion located between the first and the second conical portions; (d) at least one entry for the liquid suspension to enter the enclosure; (e) a first outlet port for collecting the clarified liquid; (f) a second outlet port to discharge the concentrated liquid suspension; and (g) a first stack of cones located within the casing. In this device, the first cone stack can occupy at least part of the first conical portion and can occupy at least part of the cylindrical portion. Each cone of the first pile of cones includes (i) a truncated apex positioned distal to the second conical portion, and (ii) an open base positioned close to the second conical portion. Optionally, the cones of the first stack are generally centered around a substantially central opening formed by the truncated apex in each cone in the first stack of cones.

[0042] An angle of inclination to the surface of a cone in the first pile of cones can vary between about 15 degrees to about 75 degrees in relation to the vertical. For example, a cross section of the cone's surface defines an arcuate line. These cones can have a convex or concave surface. In other embodiments, the angle of inclination of the cones is constant and can be, for example, about 45 degrees. The cones of the first cone stack are preferably substantially uniform in spacing.

[0043] An angle of inclination to the surface of a cone in the second pile of cones can vary between about 15 degrees to about 75 degrees in relation to the vertical. In another embodiment, the angle of inclination of the cones in the second pile of cones is about 45 degrees.

[0044] Another aspect of the present invention consists of a particle sedimentation device, which comprises: (A) an investment; (B) at least two conical plates arranged inside the enclosure; (C) a first opening in the housing; and (D) a second opening in the housing. In one embodiment, at least two conical plates are stacked on top of each other. Preferably, the housing includes between about 3 and about 30 conical plates. At least two conical plates can be separated by a substantially constant distance. Optionally, the width of a channel formed between the adjacent surfaces of at least two conical plates is between about 1 mm and about 50 mm. Three or more supports can hold each of the conical plates in the stack.

[0045] Each of the conical plates can include a truncated apex close to the first opening and an open base positioned distal to the first opening. The conical plates can generally be centered in the enclosure and are arranged in a substantially vertical position within the enclosure. The angle of inclination to the surface of each of at least two conical plates can vary between about 15 degrees and about 75 degrees in relation to the vertical. The conical plates can have a concave shape in relation to a longitudinal axis. The conical plates can have a convex shape in relation to the longitudinal axis. Therefore, the cross section of a conical plate can define an arcuate line. In one embodiment, the conical plates have a shape defined by at least one radius of curvature.

[0046] In these devices, the housing includes a cylindrical portion and a conical portion. The first opening can be associated with the conical portion. Optionally, the second opening can be associated with the cylindrical portion. The second opening can be positioned on the side wall of the cylindrical portion. The second opening can be positioned in a lid associated with an open end of the cylindrical portion. The second opening can be positioned in the conical portion.

[0047] Another aspect of the present invention consists of a sedimentation device that includes, but is not limited to: (1) an upper housing that includes: a first conical portion; a first cylindrical portion; and at least one door; (2) a lower housing interconnected to the upper housing and which includes: a second conical portion; a second cylindrical portion; and at least one door; and (3) a pile of cones located within the sedimentation device, and each cone in the cone pile includes a small opening oriented towards the first conical portion and a large opening oriented towards the second conical portion, and the first pile of cones generally centered around a longitudinal axis of the sedimentation device. Optionally, the upper housing further comprises a first flange configured to couple a second lower housing flange. The upper housing can be permanently attached to the lower housing.

[0048] In these devices, the surface of a cone of the first pile of cones is at an angle between about 15 degrees to about 85 degrees in relation to the longitudinal axis. Optionally, the first and second conical portions are concave inwards towards the longitudinal axis. In one embodiment, a longitudinal cross-section of the body of a cone forms a line with an arcuate shape.

[0049] Additionally or alternatively, the first conical portion is concave inward towards the longitudinal axis and the second conical portion is concave outwardly away from the longitudinal axis. In one embodiment, the sedimentation device includes a second stack of cones located within the sedimentation device. In another embodiment, each cone in the second pile of cones includes a small opening oriented away from the first conical portion and a large opening oriented towards the first conical portion. Optionally, the cones in the second pile of cones have bodies that are concave outwards, moving away from the longitudinal axis.

[0050] In any of the sedimentation devices of the present invention, the casing and / or the cones and / or any other components of the device may be composed of a metal or a plastic. Plastic can be one or more of polypropylene, polyethylene, polycarbonate, polystyrene and others. In one embodiment, the sedimentation device is formed entirely of plastic. In another embodiment, at least one cone in the cone stack is at least partially made of stainless steel. Metal surfaces (especially stainless steel) can be electropolished to provide a smooth surface. Likewise, in any of the sedimentation devices of the present invention, the casing and / or cones and / or any other components of the device may be totally or partially coated with one or more of a non-sticky plastic, such as Teflon or silicone.

[0051] In any of the sedimentation devices of the present invention, the casing may also include a fluid jacket associated with one or more of the first conical portion, the second conical portion and / or the cylindrical portion. In one embodiment, the fluid jacket is associated with the second conical portion and the cylindrical portion. The fluid jacket can include at least one port for receiving the fluid at a predetermined temperature. Optionally, the fluid jacket can include a second port for extracting fluid from the fluid jacket. Water or other fluids can be directed to the fluid layer to keep the cyclone enclosure and all of its contents within a desired temperature range. Doors can be formed on the outer wall of the cyclone shell to reach the jacket. The doors can act as entry or exit doors for the circulation of cooling or heating fluids through the jacket.

[0052] In any of the sedimentation devices of this description, one or more sensors can be positioned to monitor the physical conditions inside the sedimentation device. In addition, or alternatively, at least one sensor can be positioned to monitor conditions within a pipeline line interconnected to the sedimentation devices of the present description. The pipeline may be a return line interconnected to a lower outlet port on the sedimentation device.

[0053] These sensors can be selected to determine one or more of the pH, dissolved oxygen (OD), glucose, temperature and CO, »(including CO,» dissolved, known as partial CO » ) inside the enclosure or pipeline. The sensors can include one or more probes in contact with a solution within the enclosure or pipeline. The probe can be attached to an interior surface of the sedimentation device or the pipeline. In preferred embodiments, at least one sensor and / or a probe are positioned within the lower conical portion of the sedimentation device, and they can be spaced one or more between the side door and the bottom door.

[0054] These probes can transmit data without contact with a reader. In this way, the probe can measure a condition inside the sedimentation device and / or the line and transmit the data to the reader outside the sedimentation device. One or more of the probes can be a fluorescent probe. One or more of the pH, OD, glucose, temperature and pCO> can be measured by the probe inside the cyclone shell. The probe can be attached to a portion of the housing. The housing portion can be operable in order to transmit the light produced by the fluorescent probe. As described above, a portion of the wrapper can be transparent or translucent. The reader (or meter) receives the light from the fluorescent probe. The reader can also include an optical fiber that collects the light transmitted by the fluorescent probe. The appropriate probes and readers are available from several vendors, including PreSens Precision Sensing GmbH. In another configuration, the probe inside the sedimentation device can transmit data to the reader outside the sedimentation device over a network connection. For example, the probe can communicate with the reader via WiFi, Bluetooth or any other wireless communication mode.

[0055] In the operation of a sedimentation device of the present invention, the data from these sensor (s) can be used to adjust a fluid temperature inside the fluid jacket. In another embodiment, the sensor data can be used to adjust one or more of pH, temperature, dissolved oxygen concentration, dissolved carbon dioxide and nutrient concentrations within the particle sedimentation device.

[0056] Another aspect of the present invention features methods of settling particles or cells in a liquid suspension. The method includes, but is not limited to, (i) introducing a liquid suspension of the particles into a particle sedimentation device of the present invention; (ii) collecting the particles from a first opening in a sedimentation device enclosure; and (iii) the collection of a liquid from another opening in the sedimentation device. The liquid can be collected from an opening in a closure that covers at least a portion of the enclosure at an end of the enclosure opposite the first opening. The liquid can be collected from at least one additional opening in the housing, and the opening is configured to open on one side of the housing. In these methods, the step of introducing a liquid suspension into the sedimentation device may include directing a liquid suspension from a plastic bag from the bioreactor to the particle sedimentation device.

[0057] A related aspect of the present description presents a method of settling particles into a suspension. The method includes one or more of: (a) introducing a liquid | suspension of particles into a sedimentation device that includes: (i) an upper shell with a first conical portion, a first cylindrical portion and at least least one door; (ii) a lower housing interconnected to the upper housing and which includes a second conical portion, a second cylindrical portion and at least one door; and (iii) a pile of cones located within the sedimentation device, and each cone in the cone pile includes a small opening oriented towards the first conical portion and which has bodies that are concave inward towards a longitudinal axis of the device sedimentation; (b) the collection of a clarified liquid from at least one door of the

superior profit; and (c) collecting a concentrated liquid suspension from at least one port of the lower casing.

[0058] In these methods, the clarified liquid may include a first subset of particles in the suspension. The first subset of particles can comprise cell residues, dead cells and others.

[0059] The concentrated liquid can also include a second subset of particles from the suspension, and the second subset of particles can include living cells. The particles of the second subset of particles may be larger than the particles of the first subset of particles. Each particle of the second subset of particles can have a greater mass than the particles of the first subset of particles.

[0060] In these methods, a second stack of cones can optionally be positioned inside the sedimentation device. These cones that form the second pile of cones may have bodies that are concave outwardly away from the longitudinal axis of the sedimentation device.

[0061] In any of these methods, the liquid suspension may include at least one recombinant cell suspension, an alcoholic fermentation, a precipitation protein solution, a mixture of cells containing aqueous fluids and an organic layer containing organic products extracts produced by the cells, a suspension of solid catalyst particles in a liquid mixture that mostly contains depleted products and reagents, a suspension of microspheres coated with protein A molecules that can bind the monoclonal antibodies in the cell culture, a suspension of microcarrier granules with mammalian cells growing attached to the granules, municipal wastewater and industrial wastewater. In these methods, the liquid suspension can include at least one among mammalian cells, bacterial cells, yeast cells, plant cells, algae cells, human stem cells or differentiated human cells and / or insect cells. In these methods, the liquid suspension can include at least one of algae cells for biodiesel production, recombinant mammalian cells and / or hybridoma cells, yeast cells metabolically designed for the production of secreted organic products and yeast in the beer. In these methods, the liquid suspension may include the matching microbial cells selected from Pichia pastoris, Saccharomyces ce- reviae, Kluyveromyces lactis, Aspergillus niger, Escherichia coli and Bacillus-subtilis.

[0062] In these methods, the liquid collected from the sedimentation device can include at least one among biological molecules, organic or inorganic compounds, chemical reagents and chemical reaction products. In these methods, the liquid collected from the sedimentation device includes at least one among hydrocarbons, polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates, antibodies, antibodies, terpenes, isoprenoids, biodiesel, polyprenoids and beer. In these methods, the liquid collected from the sedimentation device includes at least one among biodiesel components, secreted therapeutic proteins or hormones, such as insulin or its analogs, antibodies, monoclonal antibodies, growth factors, subunits of vaccines, viruses, virus-like particles, colony-stimulating factors, erythropoietin (EPO), secreted flavor or fragrance compounds, including geraniol, mycrene, brazzein sweetening protein, etc.

[0063] In these methods, the sedimentation devices of the present invention can function as an autonomous perfusion bioreactor for the in vitro expansion of mammalian cells, such as stem cells and CAR-T cells for the therapy of autologous cells. In these examples of the sedimentation devices of the present invention, the entry of a cell culture medium free of serum or free of animal protein can be pumped continuously into the sedimentation / perfusion bioreactor through the lower port and / or the side port. A controlled mixture of O2, CO? and N> can also be pumped to control the pH and DO of the culture supernatant inside the pelletizer / bioreactor. In the end in cell expansion in vitro, the collection of sedimented cells concentrated at the bottom can be harvested from a lower port.

[0064] The above is a simplified summary of the description which is intended to provide an understanding of some aspects of the sedimentation devices of the present invention. The present summary is neither an extensive nor an exhaustive overview of the invention and its various aspects, modalities and configurations. It is not intended to identify the key or critical elements of the description or to outline the scope of the description, but to present selected concepts of the description in a simplified way, as an introduction to the more detailed description presented below. As will be appreciated, other aspects, modalities and configurations of the description are possible using, alone or in combination, one or more of the characteristics determined above or described in detail below. As will be appreciated, other modalities are possible using, alone or in combination, one or more of the characteristics indicated above or described in this document. For example, it should be considered that the various features and devices shown and / or described with respect to one modality can be combined or replaced with features or devices of other modalities, regardless of whether such a combination or substitution is or is not. shown or described specifically in this document. The-

Additional aspects of the present invention will be more evident with the Detailed Description, in particular when considered together with the drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0065] Figure 1 is a front perspective view of a sedimentation device of another configuration of the present invention.

[0066] Figure 2 is a partial sectional front view of the sedimentation device of Figure 1 illustrating a stack of convex cones within the sedimentation device.

[0067] Figure 3 is another partial sectional front view of the sedimentation device of Figure 2.

[0068] Figure 4 is an exploded front perspective view of the sedimentation device of Figure 1.

[0069] Figure 5A is a perspective view of a housing of the sedimentation device of Figure 1.

[0070] Figure 5B is a top plan view of the housing of Figure 5A.

[0071] Figure 5C is a side elevation view of the housing of Figure 5A.

[0072] Figure 6 is a top plan view of the sedimentation device of Figure 1.

[0073] Figure 7 is a front elevation cross-sectional view of the sedimentation device considered along line 7-7 of Figure 6 with the stack of cones removed for clarity.

[0074] Figure 8A is a front elevation view of detailed cross section of a portion of Figure 7.

[0075] Figure 8B is another front elevation view of detailed cross section of a portion of Figure 7.

[0076] Figure 9A is a top plan view of a cone of the sedimentation device of Figure 1.

[0077] Figure 9B is a bottom plan view of the cone of the sedimentation device in Figure 1.

[0078] Figure 9C is a side elevation view of the cone of the sedimentation device of Figure 1.

[0079] Figure 9D is a side elevation cross-sectional view of the cone of the sedimentation device considered along line 9D-9D of Figure 9A.

[0080] Figure 10 is a front perspective view of a sedimentation device of another configuration of the present description.

[0081] Figure 11 is a partial sectional front elevation view of the sedimentation device of Figure 10.

[0082] Figure 12 is another partial sectional front view of the sedimentation device of Figure 10, illustrating an upper stack of cones and a lower stack of cones within the sedimentation device.

[0083] Figure 13 is a perspective view of partial cross-section of a lower housing of the sedimentation device of Figure 10 and showing the lower stack of cones.

[0084] Figures 14 and 15 are seen from the bottom cones of the sedimentation device in Figure 10.

[0085] Figure 16 is a front perspective view of a sedimentation device of another configuration of the present description with the internal elements of the sedimentation device illustrated in discontinuous lines.

[0086] Figure 17 is a front elevation cross-sectional view of the sedimentation device of Figure 16.

[0087] Figure 18 is an exploded front perspective view of the sedimentation device of Figure 16, illustrating a second con-

optional cones adapted to be positioned inside the sedimentation device.

[0088] Figures 19A and 19B are seen in perspective of the cones of an embodiment of the present invention configured for use with the sedimentation device of Figure 16.

[0089] Figures 20A and 20B are seen in perspective of an optional conduit for use with the sedimentation devices of the present invention.

[0090] Figures 21A and 21B are seen in perspective that generally illustrate a diffuser of a modality of the present invention that is configured for use with sedimentation devices.

[0091] Figure 22 is a front perspective view of another sedimentation device of one embodiment of the present description and illustrates some internal elements of the sedimentation device in discontinuous lines.

[0092] Figure 23 is a front elevation cross-sectional view of the sedimentation device of Figure 22.

[0093] Figure 24 is a schematic representation of the attachment of a compact cell / particle sedimentation device of the present invention to a modular bioreactor.

[0094] Figure 25 is a graph showing the results of culturing the yeast cell perfusion bioreactor P. pastoris, with a compact cell sedimentator completely compacted as a cell retention device and configured as shown in Figure 24; and

[0095] Figure 26 shows the analysis of the particle size of the samples taken from the bioreactor and effluent from the setter's sedimenter as shown in Figure 24.

DETAILED DESCRIPTION

[0096] The term "one" or "one" entity refers to one or more of those entities. Accordingly, the terms "one" (or "one"), "one or more" and "at least one" can be used interchangeably in this document. The terms "comprises", "includes" and "has" can be used interchangeably.

[0097] The expressions "at least one", "one or more" and "and / or" are the expressions of open sense that are of both conjunctive and disjunctive operation. For example, each of the expressions "at least one from A, B and C", "at least one from A, B or C", "one or more from A, B and C", "one or more from A, B or C "and" A, B and / or C "means only A, only B, only C, A and B together, A and C together, B and C together, or A, Be C together.

[0098] The transitional term "comprises" is synonymous with "includes", "contains", or "characterized by" is inclusive or open-ended and does not exclude additional elements or steps of the method that are not recited.

[0099] The transitional expression "consists of" excludes any element, step or ingredient not specified in the claim, but does not exclude additional components or steps that are not related to the invention, such as the impurities ordinarily associated with them.

[0100] The transitional phrase "essentially consists of" limits the scope of a claim to the specified materials or steps or steps and those that do not materially affect the basic and new characteristic (s) (s) of the claimed invention.

[0101] With reference now to Figure 1, a configuration of a sedimentation device 300 of the present invention, useful for the sedimentation of particles or cells, is illustrated. The settling device 300 generally includes an upper housing 301A and a lower housing 301B. In one embodiment, the upper and lower shells 301A, 301B are substantially identical. Therefore, in one embodiment, housings 301A, 301B are generally interchangeable.

viable.

[0102] With reference now to Figures 2 to 9, housings 301A, 301B generally include a tapered portion 303A, 303B, a cylindrical portion 308A, 308Bb, a first port 353A, 353B and a second port 354A, 354B.

[0103] Optionally, the first door 353 is generally aligned concentric with a longitudinal axis of the housing 301. The first door 353 can be used as an input as well as an output. In exemplary embodiments, the second port 354 extends through the conical portion 303. The second port 354 can also be used for the introduction or removal of liquids, gases and solids from the sedimentation device 300. Optionally, the second door 354 can generally be aligned parallel to the longitudinal axis 350 of the cell sedimentation device. In exemplary embodiments, the second door 354 can extend through the cylindrical portion 308. Other configurations of the first and second doors 353, 354 are contemplated. Enclosure 301 can also have more than two ports. Ports 353, 354 are configured to interconnect with a pipeline.

[0104] Such a pipeline can be interconnected to any of the compact cell sedimentation devices of the present invention. The line can have a diameter or be configured to interconnect with any port of the modalities of the present invention. The line can optionally include at least one sensor positioned within a hollow interior. The sensors can come in contact with fluids and / or particles within the line. Optionally, the sensors can be arranged on an internal surface of the line, although other configurations are contemplated. The sensors can be operable to monitor one or more of the pH, DO, glucose, temperature and CO, »(including CO,» dissolved or partial) in the line. Optionally, one or more of the sensors may comprise a fluorescent probe that emits light that varies based on a condition detected by the probe. The light can be collected by a reader or a meter. Optionally, the light can be collected by an optional fiber cable and transmitted to the meter. The meter is operable to report or indicate the levels of at least one of the pH, DO, glucose, temperature and CO, detected by the fluorescent probes. The pipeline may comprise a material that is transparent or at least translucent. In this way, the light generated by a sensor can pass through the line. Alternatively, at least a portion of a line is transparent or translucent, similar to a window. Therefore, the light generated by a sensor can be transmitted through the window portion and collected by the meter.

[0105] The cones 309 can be positioned inside the sedimentation device 300. As illustrated in Figures 2 and 3, the cones 309 can be arranged in a stack with an open apex 342 facing the first port 353A. upper housing 301A and a base or large opening 346 oriented towards the first port 353A of the lower housing 301B. In exemplary embodiments, between three and twenty-five cones 309 are arranged in a stack within the sedimentation device 300. However, the casings 301 can be sized to receive any number of cones 309 when the sedimentation device 300 is mounted as illustrated in the figure.

[0106] The elements of the sedimentation device 300, such as housings 301 and cones 309, can be made of disposable plastic for a single use. Alternatively, one or more of the housings 301 and the cones 309 can be manufactured from a metal, such as a stainless steel alloy, or glass. The cone surfaces 309, and the inner surfaces of the shells 301 can be totally or partially coated with one or more of a non-sticky plastic, a Teflon, a silicone and similar materials known to those skilled in the art. In addition, or alternatively, surfaces (especially when made of stainless steel) can be electropolished to obtain a smooth surface. These sedimentation devices can be easily sized to any desired size.

[0107] Casings 301 may optionally include a fluid jacket (not shown). The fluid jacket can operate in such a way that water or other liquids can be directed to the fluid jacket through one or more ports to keep the shells 301 and the contents inside the sedimentation device 300 within a temperature range desired.

[0108] With reference now to Figures 5A to 5C, a plurality of spacers 315 can project into an internal surface of the housings 301. The spacers 315 are configured to prevent the stack of cones 309 residing within sedimentation device 300 is supported against the inner surface of shells 301A, 301B. Optionally, the spacers 315 can be more or less parallel to the longitudinal axis 350 of the sedimentation device 300. Other configurations of the spacers 315 are contemplated. Spacers 315 have a substantially thin cross section to prevent or minimize interference with the movement or flow of liquid and suspended particles within the settling device 300.

[0109] With reference now to Figure 7, spacers 315 can include a plurality of first spacers 315A, second spacers 315B and third spacers 315C. As illustrated in general, in one embodiment, each of the first 315A spacers extends over at least a portion of a surface.

inner surface of cylindrical portion 308. Second spacers 315B extend from an inner surface of conical portion 303 close to cylindrical portion 308. Third spacers 315C can be separated from second spacers 315B. Specifically, in one embodiment, the third spacers 315C are arranged closer to the first port 353 than to the cylindrical portion 303.

[0110] In one embodiment, the upper housing 301A and the lower housing 301B are fixedly joined. For example, the top and bottom housings 301 can be glued, hot welded, or sonicated to each other.

[0111] Alternatively, and with respect to Figure 1 again, optionally a flange 318 may extend from the generally cylindrical portion 308 of the shells 301. In exemplary embodiments, the flange extends more or less perpendicular to the longitudinal axis

350. The optional flange 318A is configured to interconnect with the upper housing 301A to a flange 318B of the lower housing 301B. Flanges 318A, 318B can optionally include the projections 320 which are best seen in Figure SA. In exemplary embodiments, a fastener or hook 322 is formed at a free end of each projection 320.

[0112] At least one protuberance 324 can also be formed on the flange 318. The protuberance 324 can have a shape that is generally cylindrical. The protrusion 324 is adapted to be received in a corresponding recess 326 of another flange. Additionally, or alternatively, flange 318 can include elements 332, 334 adapted to align the upper and lower shells 301A, 301B. In exemplary embodiments, the elements comprise the flaps 332 and the associated depressions 334. As illustrated in Figure 1, when the upper and lower housings 301A, 301B are aligned, the flaps 332 fit into the depressions 334 on an opposite flange.

[0113] Optionally, the flange protrusion 324 and the recess 326 can include holes. The protrusion and recess holes are configured to align when a protrusion 324 of an upper casing 301A is received in a recess 326 of a lower casing 301B (as shown in Figure 8A). In this way, an accessory 328, such as a screw, can pass through the aligned holes. A nut 330 can then be interconnected to accessory 328 to lock housings 301A, 301B to each other in a release way. As generally illustrated in Figure 8B, the projections 320 of the flange 318 are configured to lock when an upper housing 301A is aligned with a lower housing 301B. Specifically, in one embodiment, the hooks 322 of the projections 320 lock releasably.

[0114] A groove 336 can be formed on the optional flange 318. The groove 336 is configured to retain a washer or gasket 338 positioned between the upper and lower housings 301A, 301B as illustrated in general in Figures 8A and 8B .

[0115] In one embodiment, the tapered portion 303 of the shells 301 is not linear. More specifically, the conical portion 303 tapers along an arcuate passage of a maximum diameter close to the cylindrical portion 308 to a minimum diameter close to the first door.

353. More specifically, and with reference now to Figures 5C and 7, a longitudinal cross section of the tapered portion 303 of housing 301 defines a line with an arched shape between the cylindrical portion 308 and the first port 353. In one embodiment, the conical portion 303 is concave inwardly towards a center of the sedimentation device 300. In another embodiment, the conical portion 303 may have a constant radius of curvature. Optionally, in another mode, the conical portion 303 may have two or more radii of curvature.

frog. For example, the tapered portion 303 may have a first radius of curvature near the cylindrical portion 308 and a second radius of curvature near the first port 353. The central points of the first and second radii of curvature are positioned within an interior of the wrapper. Optionally, the inclination of the conical portion 308 can vary between about 15º and about 85º in relation to the longitudinal axis

350. In one embodiment, the conical portion 303 includes a convex portion close to the first port 353. The convex portion has a radius of curvature with a central point that is outside the housing.

[0116] With reference now to Figures 9A to 9D, cones 309 generally include a body 340 which has an apex 342 with a small opening 344 and a base with a large opening 346. Optionally, each of cones is formed separately. In exemplary modalities, the cones are substantially the same size and shape.

[0117] In some embodiments, the body 340 may not be linear between the small and large openings 344, 346. As illustrated in Figure 9D, a longitudinal cross section of the body 340 forms an arc-shaped line. The arcuate shape of each cone 309 can be more or less the same as the conical portion 303 of housing 301.

[0118] In some embodiments, the body 340 is concave inwards towards the longitudinal axis 350. In this way, a line drawn from a point in the large opening 346 to a point in the small opening 344 is inside an interior of the body.

[0119] Optionally, the body 340 has a constant radius of curvature. Alternatively, the body may have two or more radii of curvature. In this way, the body may have a first radius of curvature next to the small opening 344 and a second radius of curvature near the large opening 346. The central points of the first and second radii of curvature are positioned within an interior of the cone 309 In this way, a portion of the body 340 near the small opening 344 may have an inclination that is different than an inclination of the body near the large opening. For example, near the small opening 344, the body can be aligned at an angle of at least about 40 degrees to the longitudinal axis 350. On the other hand, near the large opening 346, the body can be closer to the vertical (or closer to the longitudinal axis). More specifically, the body can be tilted at an angle less than about 45 degrees from the longitudinal axis at a point close to the large opening

346. Optionally, the inclination of the body 340 can vary between about 5 and about 85 degrees in relation to the longitudinal axis.

[0120] As shown in Figures 9B and 9D, each cone 309 can include projections 313 configured to contact an adjacent cone to secure each successive cone 309 in a pile of cones at substantially equal spacing. In one embodiment, the projections 313 extend into an internal surface of the body 340. The projections 313 are configured to contact an external surface of a body 340 of an adjacent cone. Alternatively, the projections 313 may extend from an outer surface of the body 340.

[0121] The projections 313 can be dimensioned to provide any desired spacing between adjacent cones. Optionally, the projections 313 are configured to separate the adjacent cones by a distance between about 1 mm and about 2.5 cm. In exemplary embodiments, each cone 309 includes at least three projections 313.

[0122] With reference now to Figures 2 and 3, when the cones 309 are positioned inside the upper housing 301A, the body 340 of the lower cone 309A is supported by the second spacers 315B of the lower housing 301B. At least the conical portion 303 of the lower housing 301B and parts of the cylindrical portions 308A, 308B may be free of any cone. Therefore, the cells in the culture can be retained in the sedimentation device 300.

[0123] During the operation of the sedimentation device 300 of the modalities shown in Figures 1 to 9D, the cell culture medium free of serum or free of animal protein can be pumped into the sedimentation device 300 through one or more of the first - door and the second door 353, 354 of the lower housing 301B. The cell culture medium can be pumped continuously or periodically in the sedimentation device 300. Specifically, the sedimentation device 300 can operate in a batch or continuous operation.

[0124] A controlled mixture of O2, CO? and No can also be pumped into the sedimentation device 300 to control the pl and OD of the culture supernatant within the sedimentation device

300. Optionally, one or more of the second ports 354A, 354B and the first 353B of the bottom shell 301B can be used to sample the contents of the bioreactor, for example, to check the viability of the cells, and to continuously measure the pH of the liquid and the OD for inputs to a computer controlled multi-gas mass flow controller.

[0125] At the end of the cell expansion in vitro, the concentrated sedimented cells that are collected at the bottom of the sedimentation device 300 within a lower housing 301B can be harvested from the first port 353B of the lower housing. The clarified culture fluid containing any metabolic waste products, such as ammonia and lactate, or gases, along with any minor dead cells and cells residues not yet sedimented, can be removed through the first port 353A of the upper shell 301A.

[0126] Optionally, the sedimentation device 300 can be used as a combination of autonomous cell sorter / bioreactor. The growth media can be added to the cell sedimentation device via one or more of the first and second ports 353, 354. Therefore, the sedimentation device 300 can be used without an perfusion bioreactor.

[0127] In one embodiment, sensors can be positioned inside the sedimentation device 300. Optionally, the sensors can be arranged on an internal surface of one or more of the housings 301A, 301B. At least a portion of the shells 301 may comprise a plastic. In exemplary embodiments, the entire enclosure can be made up of plastic. In exemplary modalities, plastic is transparent or at least translucent. Optionally, at least a portion of the casing 301 is transparent or translucent. For example, a transparent or translucent material can be interconnected to an opening in housing 301 similar to a window. The transparent portion may comprise glass, plastic, or any other suitable material. The transparent portion may be formed of a material that is transparent in light of a predetermined band or bands of wavelengths.

[0128] When present, the sensors are positioned to contact the media within the sedimentation device

300. The sensors can be operable to monitor one or more of the pH, DO, glucose, temperature and CO, »(including dissolved or partial CO?) In the sedimentation device 300.

[0129] Optionally, one or more of the sensors may comprise a fluorescent probe operable to emit a light that varies based on a condition detected by the fluorescent probe. The fluorescent probes can be arranged in a variety of different positions within the 300 sedimentation device.

specifically, fluorescent probes can be arranged to measure different conditions, or changes in conditions, in different areas within the cell sedimentation device. Optionally, at least one fluorescent probe is affixed to an internal surface of the conical portion 303B of the lower housing 301B.

[0130] The light emitted by the fluorescent probes passes through the surface of the casing 301 (or a transparent portion of the casing) and can be collected by a reader or a meter. As described in this document, the meter is operable to report or indicate the levels of at least one of the pH, DO, glucose, temperature and CO, detected by the fluorescent probes inside the sedimentation device 300. Optionally, the light emitted by a fluorescent probe can be collected by an optional fiber cable and transmitted to the meter.

[0131] With reference now to Figures 10 to 15, another configuration of a sedimentation device 400 of the present invention, useful for the sedimentation of cells or particles, is illustrated. The sedimentation device 400 generally includes an upper housing 301 and a lower housing 401. The upper housing 301 includes a first stack of cones 309 and the lower housing 401 includes a second stack of cones 409. The upper housing 301 and cones 309 are the same, or similar, to shells 301 and cones 309 described together with Figures 1 to 9D.

[0132] The bottom housing 401 generally includes a tapered portion 403, a cylindrical portion 408, a first port 453 and a second port 454. Ports 453, 454 are configured for interconnection with a pipeline .

[0133] In one embodiment, the lower housing 401 is fixedly joined to the upper housing 301. For example, the lower housing and the upper housing can be welded (including hot-soldering), glued together, or joined by other means known to the elements skilled in the art.

[0134] Alternatively, the lower housing 401 may optionally include a flange 418. The optional flange 418 is configured to interconnect reliably with an optional flange 318 of the housing 301. Therefore, the flange 418 may include projections hooked, protrusions, recesses, flaps and depressions that work similarly to the characteristics of flange 318. Optionally, spacers 415 can extend into the cylindrical portion

408.

[0135] With reference now to Figure 13, the tapered portion 403 of housing 401 is convex inward toward longitudinal axis 450. Specifically, a straight line drawn from a point on the tapered portion near the first port 453 to a point where the conical portion intercepts the cylindrical portion 408 will be outside the housing 401.

[0136] The tapered portion 403 may have a constant radius of curvature. Alternatively, the tapered portion 403 may have two or more radii of curvature. For example, the tapered portion 403 may have a first radius of curvature near the cylindrical portion 408 and a second radius of curvature near the first port 453. The central points of the first and second radii of curvature are positioned outside the enclosure.

401. In one embodiment, the tapered portion 403 is inclined at an angle less than about 45 degrees from the longitudinal axis 450 at a point close to the first port 453. Optionally, at a point close to the cylindrical portion 408 , the conical portion has an inclination greater than about 45 degrees in relation to the longitudinal axis. In another embodiment, the inclination of the tapered portion 403 may vary between about 15 and about 85 degrees with respect to the longitudinal axis.

[0137] In exemplary embodiments, sensors can be positioned inside the sedimentation device 400. The sensors can be arranged on an internal surface of one or more of the enclosures 301, 401. The sensors can be arranged to contact the media inside sedimentation device 400. The sensors are operable to monitor one or more of the pH, DO, glucose, temperature and CO, (including dissolved or partial CO) in the sedimentation device 400. The sensors can be the same as the other sensors described in this document. Accordingly, one or more of the sensors may comprise a fluorescent probe operable to emit light that varies based on a condition detected by the fluorescent probe. The light can be transmitted through a transparent portion of the housings 301, 401 or through a window in the housings.

[0138] As shown in Figures 12 and 13, cones 409 are stacked in the lower housing 401. Cones 409 are oriented with a small opening 444 positioned close to the first door

453. The body 440 of each cone 409 has a shape that generally corresponds to the shape of the tapered portion 403 of the housing. Specifically, the cone body 440 may have an arched shape that corresponds to at least part of the conical portion of the housing. In exemplary embodiments, the cone bodies are convex inward towards the longitudinal axis 450. Optionally, the cone bodies have a constant radius of curvature. Alternatively, the cone bodies can have two or more radii of curvature. In a modality, the inclination of the bodies can vary between about 5 and about 85 degrees in relation to the longitudinal axis.

[0139] The projections 413 can be formed in the body 440 of the joint in such a way that the adjacent cones are separated by a predetermined distance. In one embodiment, the projections 413 extend into an internal surface of the cone body.

Additionally, or alternatively, the projections 413 can optionally be formed on an external surface of the cone body. When the cones are stacked together, the projections 413 contact an inner surface of a lower cone in such a way that the adjacent cones are separated by a predetermined distance. The projections 413 of the lower cone 409A will contact an internal surface of the tapered portion 403 when the cones are positioned in the housing 401. A higher cone 409E can optionally include the projections 448 that extend beyond the large opening 446. As shown in Figure 12, the projections 448 of the upper cone 409E may contact an inner surface of the lower cone 309A of the upper stack of cones 309. Contact between the projections 448 and cone 309A prevents unintentional or inadvertent movement of the stack of 409 cones.

[0140] As shown in Figures 14 and 15, in some modes, cones 409 have different diameters. A lower cone 409A may have a diameter that is larger than those of other cones in the stack. Each cone 409B to 409E can have a successively smaller diameter with the higher cone 409E which has the smallest diameter. In one embodiment, six cones 409A to 409E can be stacked in the bottom shell 401. In another embodiment, a stack of cones 409 in the bottom shell can include four to ten cones.

[0141] The sedimentation device 400, including shells 301, 401 and cones 309, 409, can be formed from the same materials as other embodiments described in this document. In exemplary embodiments, one or more of the casings and cones are made of single-use disposable plastic. Alternatively, one or more of the casings and cones are manufactured from a metal, such as a stainless steel alloy, or glass. The surfaces of the cones 309, 409 and the inner surfaces of the shells 301, 401 can be totally or partially coated with one or more of a non-sticky plastic, a Teflon, a silicone and similar materials known from the elements known in the prior art . The surfaces of the settling device 400 (especially when formed of stainless steel) can be electropolished to obtain a smooth surface. The settling device 400 can be scaled to any desired size.

[0142] The sedimentation device 400 can operate in a manner identical or similar to that of the sedimentation device

300. Specifically, the cell culture medium free of serum or free of animal protein can be pumped into the sedimentation device 400 through one or more of the first and second ports 453, 454 of the lower enclosure 401. The cell culture medium can also be pumped continuously or periodically in the sedimentation device 400. Specifically, the sedimentation device 400 can operate in a batch or continuous operation.

[0143] A controlled mixture of O2, CO? and No can also be pumped into the sedimentation device 400 to control the pH and OD of the culture supernatant within the cell sedimentation device. Optionally, one or more of the second ports 354, 454 and the first port 353 of the bottom shell 301 can be used to sample the contents of the bioreactor, for example, to check the viability of the cells, and to continuously measure the pH of the liquid and the OD for inputs to a computer controlled multiple gas mass flow controller.

[0144] At the end of the expansion of the cells in vitro, the concentrated sedimented cells that are collected at the bottom of the sedimentation device 400 can be harvested from the first port 453 of the lower case 401. The clarified culture fluid containing any products metabolic residuals, such as ammonia and lactate, or gases,

along with any minor dead cells and cell residues not yet settled, can be removed through the first port 353 of the upper shell 301.

[0145] Optionally, the sedimentation device 400 can be used as a combination of autonomous cell bioreactor / classifier. The growth media can be added to the cell sedimentation device via one or more of the first and second ports 353, 354, 453, 454. Therefore, the sedimentation device 300 can be used without an perfusion bioreactor.

[0146] With reference now to Figures 16-21, another configuration of a sedimentation device 500 for particles or cells of the present invention is illustrated. The sedimentation device 500 includes elements that are the same as, or similar to, the sedimentation devices 300, 400 of the present invention. More specifically, the settling device 500 generally includes an upper conical portion 503A, a cylindrical portion 508 and a lower conical portion 503B that define a generally hollow interior. In a fashion, the upper and lower conical portions 503A, 503B are substantially identical. At least one stack of cones 509 is positioned within the settling device 500.

[0147] The conical portions 503A, 503B generally include a first port 553 and optionally a second port 554. Optionally, the first port 553 is substantially aligned concentric with a longitudinal axis 550 of the settling device 500. A first port 553 can be used as an input as well as an output.

[0148] Second port 554 can also be used for the introduction or removal of liquids, gases and solids from the hollow interior of sedimentation device 500. In exemplary embodiments, second port 554 extends through conical portion 503. Optionally, the second port 554 can generally be aligned parallel to the longitudinal axis 550 of the cell sedimentation device. In other embodiments, the second port 554 can extend through the cylindrical portion 508. In one embodiment, the second port 554 can be oriented transverse or perpendicular to the longitudinal axis 550. Other configurations of the first and second ports 553, 554 are contemplated . The settling device 500 can also have more than four ports.

[0149] Ports 553, 554 are configured to interconnect with a pipeline. Such a pipeline can be interconnected to any of the compact cell sedimentation devices of the present invention. The pipeline can have a diameter or be configured to interconnect with any port of the modalities of the present invention. The line can optionally include at least one sensor positioned inside a hollow interior. The sensors can come in contact with fluids and / or particles within the line. Optionally, the sensors can be stored on an internal surface of the line, although other configurations are contemplated. The sensors can be operable to monitor one or more of the pH, OD, glucose, temperature and CO, (including dissolved or partial CO) in the line.

[0150] Optionally, one or more of the sensors may comprise a fluorescent probe that emits light that varies based on a condition detected by the probe. The light can either be collected by a reader or a meter. The light can be optionally collected by an optional fiber cable and transmitted to the meter. The meter is operable to report or indicate the levels of at least one of the PH, OD, glucose, temperature and CO, detected by the fluorescent probes. The line can comprise a material that is transparent

current or at least translucent. In this way, the light generated by a sensor can pass through the line. Alternatively, at least a portion of a line is transparent or translucent, similar to a window. Therefore, the light generated by a sensor can be transmitted through the window portion and collected by the meter.

[0151] A conduit 560 can optionally be interconnected to at least one of the second ports 554 within the sedimentation device 500. One embodiment of a conduit 560 of the present invention is generally illustrated in Figures 20A and 20B. A 562 lumen extends through the conduit. In one embodiment, conduit 560 is not linear. More specifically, conduit 560 can be folded. In this way, the conduit is configured to extend inwardly inside the sedimentation device 500 with a free end 564 of the conduit positioned close to the longitudinal axis 550 as illustrated in general in Figure 17. Therefore, the lumen 562 through conduit 560 can be positioned to inject or withdraw fluid from a median portion of the sedimentation device 500, such as from the inside of a cone 509. In this way, the withdrawal of fluid from the sedimentation device 500 through of the conduit 560 can facilitate the fluid flow upwards within the sedimentation device in such a way that the cells or particles within the fluid sediment in the cones and migrate to the lower conical portion 503B.

[0152] The settling device 500 may also include a diffuser 570 as illustrated in general in Figures 21A and 21B positioned within the hollow interior. The diffuser 570 can be associated with one of the second ports, such as the second lower port 554B. The fluid can be injected or removed from the settling device 500 through the diffuser without disturbing the particles or cells that have settled near the lower conical portion 503B. When fluid is injected into the sedimentation device 500 through the diffuser, the fluid, which may contain cells or particles, is evenly distributed throughout the lower conical portion 503B of the sedimenter.

[0153] With reference now to Figures 21A and 21B, the diffuser may comprise a torus or ring 574 extending from a stem 572. The stem 572 can be generally linear and configured to be oriented parallel to the longitudinal axis 550. The ring 574 can be configured to extend around longitudinal axis 550 when diffuser 570 is interconnected to settling device 500. In one embodiment, ring 574 is adapted to be substantially concentric with the longitudinal axis.

[0154] An opening 576 is formed through ring 574 to facilitate the transport of fluid, cells or particles through the diffuser. In one embodiment, opening 576 is formed on one side of the ring connected to stem 572. In this way, opening 576 can be oriented to the first lower door 553B when the diffuser is interconnected to the second lower door 554. Opening 576 can be configured as a single channel or groove. The groove can extend substantially continuously around the ring.

[0155] Alternatively, the ring may comprise a plurality of individual openings 576. In one embodiment, the openings are oriented axially to eject the fluid generally parallel to the longitudinal axis. All openings 576 can be oriented in the same direction. Alternatively, some of the openings may face different or opposite directions. Optionally, one or more of the openings 576 can be oriented transverse to the longitudinal axis 550. Additionally, or alternatively, some of the openings can be oriented radially or axially.

[0156] With respect to Figure 17 again, cones 509 can be positioned inside the sedimentation device 500 and be oriented

designed to face one or more of the upper conical portion 503A and the lower conical portion 503B. In one embodiment, the sedimentation device includes a stack of cones with a small end or apex 542 of cones 509B oriented towards the first lower port 553B of the lower conical portion 503B. In this mode, a base or large opening 546 of cones is oriented towards the first upper door 553A of the upper conical portion 503A. In exemplary modalities, between three and twenty-five 509 cones are arranged in a pile inside the sedimentation device

500. In another embodiment, the stack includes 6 to 14 cones, or 10 cones. However, the sedimentation device 500 can be dimensioned to receive any number of cones 509 when the sedimentation device 500 is mounted as shown in Figures 16 and 17. At least part of the lower conical portion 503B may not contain no cone. More specifically, a lower cone 509 can be spaced a predetermined distance from an internal surface of the lower conical portion 503B. Therefore, the cells in the culture can be retained in the sedimentation device 500, for example, near the first lower port 553B.

[0157] When the cones 509B are oriented with their apices 542 next to the first lower door 553B, a body 540 of the lower cone 509 can be supported by the diffuser 570. More specifically, as illustrated in general in Figure 17, the lower cone 509 can extend through ring 574 of the diffuser in such a way that the body 540 of the cone contacts the ring of the diffuser. The lower cone can optionally be joined or welded to the diffuser ring. In this way, diffuser 570 is operable to position the lower cone 509 at a predetermined distance from the inner surface of the lower conical portion 503B.

[0158] With respect to Figure 17 again, optionally a flange

g and 518 may extend from a large end of the conical portions 503 of the sedimentation device. The flange 518 may have an internal diameter that is more or less the same, but larger, than the outer diameter of the cylindrical portion 508. In one embodiment, when the sedimentation device is mounted, the flange 518 extends outside an outer surface of the cylindrical portion 508 and more or less parallel to the longitudinal axis 550. The optional flange 518 is configured for interconnection with a conical portion 503 associated with the cylindrical portion 508. For example, a conical portion 503 can be welded or fixed to the cylindrical portion 508 next to the flange 518.

[0159] Additionally, or alternatively, flange 518 may include elements adapted to align an associated conical portion 503 with the cylindrical portion 508. In an exemplary embodiment, the elements comprise projections configured to engage the corresponding recesses in the cylindrical portion.

[0160] The flange can be configured to retain a washer or gasket positioned between the tapered portion and the cylindrical portion. The gasket can be identical or similar to the gasket 338 illustrated in general in Figures 8A and 8B.

[0161] In one embodiment, one or more of the conical portions 503 of the sedimentation device 500 are not linear. More specifically, the conical portions 503 can taper along an arched passage of maximum diameter close to the cylindrical portion 508 to a minimum diameter close to the first door 553. More specifically, and with respect to Figure 17 again, a longitudinal cross section of each of the conical portions 503 defines a line with an arched shape between the cylindrical portion 508 and the first port 553. In one embodiment, the conical portions 503 are concave inwardly towards a center of the sedimentation device tion 500. In another embodiment, conical portions 503 may have a constant radius of curvature. Optionally, in another mode, one or more of the 503 conical portions may have two or more radii of curvature. For example, a conical portion 503 may have a first radius of curvature close to cylindrical portion 508 and a second radius of curvature close to a first associated port.

553. The central points of the first and second radii of curvature are positioned within an interior of the sedimentation device

500. Optionally, the inclination of a conical portion 508 can vary between about 5 and about 85 degrees in relation to the longitudinal axis 550. In one embodiment, a conical portion 503 can include a convex portion near the first port 553. The convex portion has a radius of curvature with a central point that is outside the sedimentation device 500.

[0162] With reference now to Figures 19A and 19B, cones 509 generally include a body 540 that has an apex 542 with a small opening 544 and a base with a large opening 546. Optionally, each of the cones is formed separately . In exemplary modes, the cones are substantially the same size and shape.

[0163] In some embodiments, the body 540 may not be linear between the small and large openings 544, 546. As shown in general in Figure 17, a longitudinal cross section of the body 540 will form a line with a shape arched. The arched shape of each cone 509 can be more or less the same as that of one or more of the conical portions 503 of the sedimentation device 500.

[0164] In some embodiments, the body 540 is concave inward towards the longitudinal axis 550. In this way, a straight line drawn from a point in the large opening 546 to a point in the small opening 544 is inside an interior of the body .

[0165] Optionally, the body 540 has a constant radius of curvature. Alternatively, the body may have two or more radii of curvature. In this way, the body may have a first radius of curvature close to the small opening 544 and a second radius of curvature close to the large opening 546. The central points of the first and second radii of curvature are positioned within an interior of cone 509 In this way, a portion of the body 540 near the small opening 544 may have an inclination that is different from an inclination of the body near the large opening. For example, near the small opening 544, the body can be aligned at an angle of at least about 40 degrees to the longitudinal axis 550. On the other hand, near the large opening 546, the body can be closer to the vertical ( or closer to the longitudinal axis). More specifically, the body can be tilted at an angle of less than about 45 degrees from the longitudinal axis at a point close to the large opening 546. Optionally, the inclination of the body 540 can vary between about 5 and about 85 degrees from the longitudinal axis.

[0166] As shown in Figures 19A and 19B, each cone 509 can include projections 513 configured to contact an adjacent cone to substantially secure each successive cone 509 in a stack of cones at the same spacing. In one embodiment, the projections 513 extend outward from an external surface of the body 540. The projections 513 are configured to contact an internal surface of a body 540 of an adjacent cone. Alternatively, the projections 513 can extend from an internal surface of the body 340. In some embodiments, the projections 513 are generally oriented parallel to the longitudinal axis 550.

[0167] Projections 513 can be dimensioned to obtain any desired spacing between adjacent cones. Optionally, the 513 projections are configured to separate the adjacent cones by a distance between about 1 mm and about 2.5 cm. In exemplary embodiments, each cone 509 includes at least three projections 513.

[0168] Projections 513 can be optionally configured to fix a first cone in relation to a second cone. More specifically, the 513 projection can include a 532 flange and a groove

536. The groove 536 of a first cone can receive a flange 532 of an adjacent second cone as generally illustrated in Figure 19A.

[0169] With reference now to Figure 18, the settling device 500 may optionally include a second stack of cones 509A. The 509A cones in the second cone stack may be the same as the 509B cones. Alternatively, cones 509A may be of a different size or shape than cones 509B. In one embodiment, each of the 509A cones in the second cone stack may be of a different size. For example, the uppermost of the 509A connectors may have a diameter that is larger than the lowermost of the cones. Similarly, the lowest of the 509A cones may have a diameter that is smaller than the other cones in the second pile of cones.

[0170] Optionally, one or more spacers (not shown) can project into an inner surface of the sedimentation device 500. The spacers are configured to prevent the stack of cones 509 residing within the sedimentation device 500 be supported against the inner surface of the conical portions 503 or the cylindrical portion 508. Optionally, the spacers can be more or less parallel to the longitudinal axis 550 of the sedimentation device 300. The spacers can have a substantially thin cross section to prevent or minimize interference with the movement or flow of liquid particles and sustain

dressings within the sedimentation device 500. Although not shown in Figures 16 to 18, the spacers may be identical or similar to the spacers 315 shown in Figures 5A, 5Be7e described herein.

[0171] The elements of the sedimentation device 500, such as the conical portions 503, the cylindrical portion 508 and the cones 509, can be manufactured from a single-use disposable plastic. Alternatively, one or more of the conical portions 503, the cylindrical portion 508 and the cones 509 can be manufactured from a metal, such as a stainless steel alloy, or glass. The cone surfaces 509, and the internal surfaces of the conical portions 503 and the cylindrical portion 508 may be totally or partially coated with one or more of a non-sticky plastic, a Teflon, a silicone and similar materials known to the elements known in the state of technical. In addition, or alternatively, the surfaces (especially when formed of stainless steel) can be electropolished to obtain a smooth surface. These sedimentation devices can easily be scaled to any size desired.

[0172] In one embodiment, the conical portions are fixedly attached to the cylindrical portion, for example, by a weld (such as a sonic weld or a hot weld), an adhesive, or a glue. Optionally, one or more of the cones can be attached to an internal surface of the sedimentation device. For example, in one embodiment, a portion of the uppermost cone 509 in the cone stack may contact, and be attached to, an inner surface of the upper conical portion 503A as generally illustrated in Figure 17. In one embodiment, the cones can be joined together to form the pile of cones.

[0173] The sedimentation device 500 may optionally include a fluid jacket (not shown). The fluid jacket can be associated with one or more of the conical portions 503 and the cylindrical portion 508. Water or other liquids can be directed to the fluid jacket through one or more ports to maintain the sedimentation device 500 and its contents, including the fluid, within a desired temperature range.

[0174] During the operation of the sedimentation device 500 of the modalities shown in Figures 16 to 18, the cell culture medium free of serum or free of animal protein can be pumped in the sedimentation device 300 through one or more among the first and second ports 553B, 554B of the lower conical portion 503B. The cell culture medium can be pumped continuously or periodically into the sedimentation device 500. Specifically, the sedimentation device 500 can operate in a batch or continuous operation.

[0175] A controlled mixture of O2, CO? and No can also be pumped into the sedimentation device 500 to control the pl and OD of the culture supernatant within the sedimentation device

500. Optionally, one or more of the second ports 554A, 554B and the lower conical portion 503B, and the first port 553B, can be used to sample the contents of the bioreactor, for example, to check cell viability, and to continuous measurement of liquid pH and DO for inputs to a computer controlled multiple gas mass flow controller.

[0176] At the end of the in vitro cell expansion, the concentrated sedimented cells that are collected at the bottom of the sedimentation device 500 within the lower conical portion 503B can be harvested from the first port 553B of the sedimentation device 500. clarified culture containing any metabolic waste products, such as ammonia and lactate, or gases, along with any minor dead cells and cell residues not yet sedimented

can be removed via the first port 553A of the upper conical portion 503A.

[0177] Optionally, the sedimentation device 500 can be used as a combination of autonomous cell bioreactor / classifier. The growth media can be added to the cell sedimentation device via one or more of the first and second ports 553, 554. Therefore, the sedimentation device 500 can be used without a connection to a bioreactor. infusion.

[0178] In one embodiment, sensors can be positioned inside the sedimentation device 500. Optionally, the sensors can be arranged on an internal surface of one or more of the 503 conical portions and the 508 cylindrical portion. For exemplary purposes, at least a portion of the settling device 500 may comprise a plastic. In exemplary embodiments, the entire casing may be composed of plastic. In exemplifying modalities, plastic is transparent or at least translucent. Optionally, at least a portion of the settling device 500 is transparent or translucent. For example, a transparent or translucent material can be interconnected to an opening in the sedimentation device 500, similar to a window. The transparent portion may comprise glass, plastic, or any other suitable material. The transparent portion may be formed of a material that is transparent in light of a predetermined band or bands of wavelengths.

[0179] When present, the sensors are positioned to contact the media inside the sedimentation device

500. The sensors can be operable to monitor one or more of the pH, OD, glucose, temperature and CO, »(including dissolved or partial CO?) In the 500 sedimentation device.

[0180] Optionally, one or more of the sensors may comprise a fluorescent probe operable to emit light that varies based on a condition detected by the fluorescent probe. Fluorescent probes can be arranged in a variety of different positions within the sedimentation device 500. More specifically, fluorescent probes can be arranged to measure different conditions, or changes in conditions, in different areas within the sedimentation device. sedimentation of cells. Optionally, at least one fluorescent probe is affixed to an internal surface of the lower conical portion 503B of the sedimentation device.

[0181] The light emitted by the fluorescent probe passes through the surface of the sedimentation device (or a transparent portion of the sedimentation device) and can be collected by a reader or a meter. As described in this document, the meter is operable to report or indicate the levels of at least one of the PH, OD, glucose, temperature and CO, detected by the fluorescent probes inside the sedimentation device 500. Optionally- In addition, the light emitted by a fluorescent probe can be collected by an optional fiber cable and transmitted to the meter.

[0182] With reference now to Figures 22 and 23, another sedimentation device 600 of the present invention is generally illustrated. The settling device 600 is similar to the settling device 500 and includes many of the same characteristics. For example, the settling device 600 generally includes an upper conical portion 503A, a cylindrical portion 508 and a lower conical portion 503B that define a generally hollow interior. A diffuser 570 can be positioned inside the hollow interior in fluid communication with a second lower door 553B.

[0183] A stack of cones 509A can be positioned inside the sedimentation device 600. In a striking manner, cones 509A are oriented with their apex 542 close to the upper conical portion 503A and a first upper port 553A.

[0184] Cones 509A can be attached to an internal surface of the upper conical portion 503A. More specifically, in a fashion, cones include projections 513 as described in this document. The projections 513 of an upper cone 509A can be fixed or welded to an internal surface of the upper conical portion 503A as illustrated in general in Figure 23.

[0185] Optionally, a second cone stack (not shown) can be positioned inside the sedimentation device 600. The cones of the second cone stack can be oriented with their apexes close to the lower conical portion 503B. In one embodiment, the cones in the second cone stack are identical or similar to the 509A cones. Alternatively, the cones of the second cone stack may be of a different size or shape than that of the 509A cones. In one embodiment, the second cones can have successively increasing diameters similar to cones 409 illustrated in Figures 14 and 15.

[0186] In each of the embodiments of the present invention, the angle of inclination of the conical surfaces of stacked cone surfaces can be between about 30 degrees and about 60 degrees in relation to the vertical. In certain embodiments, the angle of inclination for the conical surface surfaces or the stacked cones is about 45 degrees from the vertical. In yet another embodiment, the angle of inclination varies between about 15 degrees and about 75 degrees. As described above, for the separation of stickier particles (typically mammalian cells), the angle of inclination is preferably closer to the vertical (ie, about 30 degrees from the vertical). For less sticky solid particles (e.g., catalyst particles), the angle of inclination may be further away from the vertical (preferably about 60 degrees from the vertical).

[0187] The construction material of any of the sedimentation devices of the present invention, including the casing, the legs and / or any additional components of the sedimentation device, may be stainless steel (especially 316 stainless steel). ), or similar materials used for applications in the cultivation of microbial or mammalian cells, as well as other metals used for applications in chemical process industries, such as catalyst separation and recycling. Stainless steel surfaces can be fully or partially electropolished to provide smooth surfaces on which cells or particles can slide after settling the liquid suspension. Some or all of the surfaces of the sedimentation device of the present invention can be coated with a non-sticky plastic or silicone, such as dimethyl dichloro silane. Alternatively or in addition, the material construction of any of these sedimentation devices of the present invention may be non-metals, including plastics, such as single-use disposable plastics. Although the metal sedimentation devices of the invention can be constructed by rolling standard plate and welding of angular steel plates to the bottom of the spiral plate, a plastic sedimentation device of the present invention, or the individual parts thereof, can be manufactured more easily continuously as a single piece when using, for example, injection molding or three-dimensional printing technologies.

[0188] In any of the sedimentation devices of the present invention, the liquid can be directed to, or extracted from, any of the ports or openings in the sedimentation device by one or more pumps (for example, a pump peristaltic) in liquid communication with the door or opening. Such pumps, or other means that cause the liquid to flow into or out of sedimentation devices, can operate continuously or intermittently. If they are operated intermittently, during the period when the pump is off, the sedimentation of the particles or cells occurs while the surrounding fluid is still quiescent. This allows particles or cells that have already sedimented to slide down the inclined conical surfaces unimpeded by the upward flow of liquid. Intermittent operation has the advantage that it can improve the speed at which cells slide, thereby improving cell viability and productivity. In a specific embodiment, a pump is used to direct a liquid suspension of cells from a bioreactor or a fermentation medium to the sedimentation devices of the present invention.

[0189] The thickness of the material that makes up the cones placed inside the casing of any of the sedimentation devices of the present invention is preferably as thin as necessary to maintain the rigidity of the shape and minimize the weight of the concentric stack of cones to be supported inside the enclosure. The radius and height of these devices can be increased independently as much as is necessary for large-scale processes as can be calculated from predictive equations as provided for inclined plate sedimenters (Batt et al. 1990, supra) .

[0190] An important factor causing the separation of particles in the sedimentation devices of the present invention is the enhanced sedimentation on inclined surfaces, which was successfully demonstrated by Boycott (Nature, 104: 532, 1920) with blood cells and on inclined rectangular surfaces as demonstrated by Batt et al. (1990, supra) with hybridoma cells producing monoclonal antibodies. The additional factors that enhance the separation of cells / particles are the centrifugal force in the cells.

particles / particles during their course above the annular regions between successive cylinders and sedimentation due to gravity on the sedimentation surfaces.

[0191] Although lamellar plates are used to enlarge the slanted plate settlers by each dimension independently, that is, increasing the length, or the width or the number of plates stacked on top of each plate, the zone of spiral conical sedimentation can be enlarged in three dimensions simultaneously simply by increasing the horizontal radius of this device. As the horizontal radius of the device increases, the number of vertical and tapered surfaces can be proportionally increased by maintaining a constant distance (or channel width) between successive spirals. The efficiency of particle separation is directly proportional to the total horizontal projected area of the inclined sedimentation surfaces. With an increase in the radius of the device, the projected horizontal area increases proportionally to the radius square, which results in a three-dimensional strip in the total projected area (that is, proportional to the radius cube) simply to the increase. increase the radius.

[0192] The sedimentation device 600 can operate in a manner similar to other sedimentation devices of the present invention. For example, sedimentation device 600 can be used and operated as described in conjunction with sedimentation devices 300, 400, 500. Methods of Use and Operation of Processes

[0193] The exemplary methods of using the sedimentation devices of the present invention are now described. A liquid containing particles (including, for example, cell culture liquid, waste water or reaction fluid containing solid catalyst particles, etc.) is introduced into a sedimentation device of the present invention through a port. About 50% to 99% of the inlet liquid (typically about 90%) is removed through a door at the bottom of the sedimentation device, while the remaining 1% to 50% (typically about 10%) of the liquid are removed through a port on the top of the device. A pump (such as a peristaltic pump) can be used to suck the liquid out of the upper port, while the concentrated liquid that exits from the bottom can escape through the lower outlet of the cyclone casing due to gravity, without the need of a bomb. Alternatively, the liquid containing sedimented cells or particles can be pumped out of a lower conical sediment port at about 50% to 99% flow rate of the incoming liquid, and the remaining clarified liquid (1% to 50% ) can exit through an upper door. Optionally, the fluid coming out of the port can be pumped out on a harvest line.

[0194] Most of the incoming cells (or particles) are pushed against the set walls of the sedimentation device by centrifugal forces at the entrance, sedimenting the conical portion through a gentle vortex movement initially , getting faster when the liquid and particles go down and exit through the bottom door. The cells or particles that have not sedimented will move upwards through the cone cells. As the liquid slowly moves upward through the cone cells, the larger particles (eg, living cells) will settle on the surfaces of the cones and slide through the cones or fall through the small spacing provided between the cones and the cones. outer walls of the cyclone shell. These sedimented particles fall vertically along the outer cylindrical walls until they reach the lower conical section of the assembly and continue to slide down the conical section to the lower door.

[0195] With the increased flow of liquid entering through the door, it is possible to reduce the residence time of the liquid within the sloping sedimentation zones in such a way that the smaller particles (for example, dead cells and cell residues) do not they will have sedimented by the time the liquid reaches the top of the sedimentation zone and, therefore, these smaller particles leave the sedimentation device through the upper door. This feature provides a simple method for selectively removing smaller particles (such as dead cells and cell debris) through the upper gate in a harvesting stream, while larger particles (such as living, productive cells) are returned. from the lower door to another vessel (such as a bioreactor).

[0196] Thus, in these methods, the step of introducing a liquid suspension in these sedimentation devices may include the directing of a liquid suspension from a plastic bioreactor bag to the particle sedimentation device.

[0197] The liquid can be directed to, or extracted from, any ports or openings in the sedimentation device by one or more pumps (for example, a peristaltic pump) in liquid communication with the door or opening. Such pumps, or other means that cause the liquid to flow into or out of the sedimentation devices, can operate continuously or intermittently. If they are operated intermittently, during the period when the pump is switched off, sedimentation of the particles or cells occurs when the surrounding fluid is still quiescent. This allows particles or cells that have already sedimented to slide over the inclined conical surfaces unimpeded by the upward flow of liquid. Intermittent operation has the advantage that it can improve the speed at which cells slide down, thereby improving cell viability and productivity. In a specific embodiment, a pump is used to direct a liquid suspension of the cells of a bioreactor or fermentation medium to the sedimentation devices of the present invention.

[0198] A parameter that can be adjusted in these methods of using the sedimentation devices of the present invention is the flow of liquid into and out of the sedimentation devices. The liquid flow rate will depend entirely on the particular application of the device, and the rate can be varied in order to protect the particles being sedimented and separated from the clarified liquid. Specifically, the flow may have to be adjusted to protect the viability of the living cells that can be separated in the seed devices of the present invention and returned to a cell culture, but the flow must also be adjusted to prevent the substantial accumulation of cells or particles in the sedimentation devices or the filling of the ducts that transfer the liquid into and out of the sedimentation devices.

[0199] In these methods, the clarified liquid collected from the sedimentation device can include at least one among biological molecules, organic or inorganic compounds, chemical reagents and chemical reaction products. The clarified liquid collected from the sedimentation device can include at least one among hydrocarbons, polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates, antibodies, isoprenoids, biodiesel and beer. In the examples of these methods, the clarified liquid collected from the sedimentation device includes at least one of the insulin or its analogs, monoclonal antibodies, growth factors, subunit vaccines, viruses, virus-like particles, stimulating factors colonies and erythropoietin (EPO).

[0200] Each publication or patent cited in this document is incorporated into this document for reference in its entirety. The sedimentation devices of the present invention that are now being generally described will be more readily understood by reference to the following examples, which are included merely for purposes of illustrating certain aspects of the modalities of the present invention. The examples are not intended to limit the invention, just as an element skilled in the art must recognize from the above teachings and the examples below that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the present invention.

EXAMPLES Example 1 Yeast or other microbial cells that secrete protein products

[0201] Recombinant microbial cells, such as from yeast or fungi (Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus niger, etc.) or bacteria cells (Escherichia coli, Bacillus subtilis, etc.), which were designed to secrete proteins heterologous (for example, insulin or brazein) or naturally secrete enzymes (for example A. niger, B. subtilis, etc.) can be grown in bioreactors coupled with the compact sedimentation devices of the present invention, to recycle live and productive cells back to the bioreactor, which will thus result in high cell densities and high productivity. Fresh nutrient media are continuously supplied to living, productive cells within the high cell density bioreactors and secreted proteins or enzymes are collected continuously at the clarified outlet of the upper port (or exits from the top side 353A, 354A, 553A, 554A) , while the concentrated living and productive cells are returned back to the bioreactor. Since dead cells and a small fraction

a few live cells are continuously removed from the bioreactor through the harvest outlet, cell growth and protein production can be maintained indefinitely, with no real need to complete the operation of the bioreactor. In operations using Pichia yeast cells with the conical sedimentation devices of the present invention, the perfusion bioreactor is operated for more than one month. Since the microbial cells grow in the suspension culture and the cell retention device can be sized to any desired size, a sedimenter of the present invention can be attached to the suspension bioreactors of sizes ranging from the laboratory scale (<1 liter ) on an industrial scale (> 50,000 liters) or any size between them to obtain high cell density perfusion cultures.

[0202] In a specific example, a culture of the cell perfusion bioreactor of the yeast Pichia pastoris is described. Pichia pastoris yeast cells were grown in a 5-liter computer-controlled bioreactor in batch mode to grow cells from the inoculum for the first 50 hours, then in fed batch mode to slowly fill the pelletizer of 12-liter cells for the next 100 hours, and then in continuous perfusion mode with a compact cell pelletizer of the present invention to remove the smaller dead cells and recycle the larger living cells back to the bioreactor. A typical schematic diagram of the connection of a compact cell / particle sedimenter of the present invention to any modular bioreactor is shown in Figure 24.

[0203] With respect to Figure 24, the yeast cells Pichia pastoris were grown in a perfusion bioreactor 218. The growth media was added to bioreactor 218 from the media reservoir 200 via a first 202 inter-pump.

connected to inlet line 201. The dissolved oxygen content and pH were monitored continuously in the bioreactor 218 by the dissolved oxygen monitor 206 and by the pH monitor 204. The yeast cell culture of the bioreactor 218 was passed to a cell sedimentator. - the 12 liter compact 208 of the present invention through a second pump 214 interconnected to line 212. The effluent from the compact cell sedimentator 208, which contained smaller dead cells, was evacuated by the effluent line 210. The cells Larger live samples were recycled from cell pelletizer 208 back to bioreactor 218 via third pump 216 and return line 217. Media and cell culture levels in bioreactor 218 were controlled by removing excess culture of cells through the fourth pump 220 and the removal line 222 to be captured or discarded.

[0204] The results obtained with this perfusion bioreactor configured with a compact cell sedimentator of the present invention are shown in Figure 25. The circles show the optical density of samples from the bioreactor, measured at 600 nm, formed during the initial batch culture and batch culture fed about 150 hours, followed by continuous perfusion operation of up to

1,600 hours or more than 2 months. The effluent or sediment collection rate is adjusted by manipulating the adjustment of the sediment inlet pump and / or the adjustment of the sedimentation recycling pump. The concentration of cells (as measured by the OD at 600 nm) and the size distribution are determined by the flow of the harvest and the cell size distribution of the cells entering the bioreactor and other factors such as the recycling ratio of the seeder. The effluent stream contains very small cells, as measured by very low ODs in the range of 0 to 30, even when the infusion rate is gradually increased from 2,000 ml / day to more than 6,000 ml / day. These results demonstrate that the very high density of cells was obtained and maintained in the bioreactor due to the recycling of most of the living cells back to the bioreactor and the selective removal of small dead cells and cell residues. Even at these rising perfusion rates, the bioreactor can be operated indefinitely at a high density of cells with no reason to shut down the bioreactor, such as membranes filled in competitive cell-based cell retention devices.

[0205] The samples from the bioreactor and the effluent from the sedimenter were analyzed at the same time with a particle size analyzer. The normalized results of the cell size distribution shown in Figure 26 clearly indicate that the effluent from the sedimenter contains a significantly smaller cell size distribution compared to that found for the cells in the bioreactor. These results demonstrate that the pelletizer removed the smaller dead cells and all cell residues, preferably in the effluent, while the larger living cells are preferably returned to the bioreactor. In this way, the bioreactor is cleaned continuously by the selective removal of dead cells and cell residues by the sediment effluent and consequently there is no accumulation of dead cells and cell residues inside the bioreactor, as is the case with all other cell retention devices.

[0206] Samples of effluents from the bioreactor and sedimenter from an advanced point in time during the perfusion culture were collected and centrifuged in small 2 ml flasks. The cells piled in the effluent from the sedimentation device 208 and the cells piled in the bioreactor 218 showed that the piled cells in the bioreactor occupy almost 50% of the volume of wet compacted cells in the flask, while the cells piled in the sedimentation effluent they only occupy about 5% of the volume of wet compacted cells. These results again confirm that only a very small fraction of the smallest intact cells in the bioreactor are removed in the effluent from the sedimenter while most of the larger intact cells are preferably returned to the bioreactor.

[0207] The concentrations of total protein in the effluent of the bioreactor and sedimentator during this 2-month-long infusion operation were measured and showed that after the initial batch and fed batch operation, that is, during the batch operation. prolonged melting, the total protein content in the effluent sample of the sedimentation device 208 is consistently higher than the total protein content in the sample of bioreactor 218. These results strongly suggest that there is no sifting of protein inside the sedimenter 208, as is generally observed with membrane-based cell retention devices such as ATF in mammalian cell perfusion cultures. In addition, these results suggest that there is some additional protein production in sediment 208, causing protein effluent concentrations to be consistently higher than those in bioreactor 218 at the same time.

[0208] The total accumulated protein in the collection stream of the continuous perfusion bioreactor configuration shown in Figure 24 can be compared with the protein that can be harvested in the cell-free supernatant of a single batch fed bioreactor 218 run more than 158 hours or almost 6 days, and repeated several times for the same duration of the culture, say 1,600 hours. Although fed batch cultures typically have a long downtime to harvest or empty the bioreactor,

internal profiles, steam sterilize in situ, cool, refill the bioreactor with sterile medium, inoculate the bioreactor with fresh cells and then allow the cells to grow to a cell density high enough to see a significant increase in protein titer , the continuous perfusion bioreactor continues to operate without interruption at a high cell density and a high production rate throughout the culture operation. As a result, the total accumulated protein in the continuously harvested product stream is increasing, at a significantly faster rate while the infusion rate is increased, and up to 160 g, a 5 times greater amount of protein than it can accumulate. be harvested in cell-free supernatants from 8 repeated batch culture operations fed in the same 5-liter bioreactor. Example 2 Removal of beer yeast cells

[0209] In large-scale brewing operations, yeast cells are removed from beer produced by filtration devices, which are regularly filled, or centrifugation devices, which are expensive high-speed mechanical devices. Previously, hydrocyclones have been tested in vain for this application (Yuan et al., 1996; Cilliers and Harrison, 1997). These devices can be replaced immediately by the sedimentation devices of the present invention to clarify the beer from the upper outlets and to remove the concentrated suspension of yeast cells from the lower outlets. Due to the increased residence time and the enhanced sedimentation in the conical zones of the sedimenter of the present invention, the author of the present invention obtained a successful separation of yeast cells from the cell culture liquid, harvesting the culture supernatant which contains only about 5% of the cells that enter the sedimentation device in its first operation. Since the device can be enlarged or reduced to increase or decrease its cell separation efficiency, it is possible to obtain completely cell-free beer from the harvest door, if desired. Thus, the devices of the present invention can be particularly useful in the manufacture of beer, as well as in the bleaching of beer, and in continuous brewing arrangements. Example 3 Bleaching or removing cells from mammalian cell culture broth

[0210] Similar to the example above, the clearing of mammalian cells from the cell culture broth at the end of a culture of the fed batch bioreactor is a necessary first step in harvesting the secreted product, such as antibodies or therapeutic glycoproteins , to be followed by a series of other downstream processing operations. Currently, centrifugation and deep filtration are used as common unit operations to remove mammalian cells and cell residues from the cell culture broth. However, periodic removal of accumulated cells from the continuous centrifugation process results in repeated downpouring of the cells in the clarified cell culture supernatant. The sedimentation devices of the present invention produce a continuously clarified supernatant (cell free or significantly depleted of cells) since mammalian cells are easily sedimented within the device. These compact sedimentation devices offer more consistent removal of cells from the cell culture broth, potentially replacing the need for any centrifugation and reducing the amount of membrane area required in a secondary depth filtration operation to completely eliminate any cells remnants and all cell waste. The whitening can be in discontinuous operations or in continuous operations in perfusion bioreactors as described below. Example 4 Mammalian cell perfusion cultures

[0211] Enhanced sedimentation of mouse hybridoma and recombinant mammalian cells in inclined sedimenters has already been successfully demonstrated (Batt et al., 1990 and Searles et al., 1994) and expanded in lamellar sedimenters (Thompson and Wilson, Paten - US No. 5,817,505). Although lamellar sedimenters are enlarged in three dimensions independently, a conical sedimentation device of the present invention can be enlarged in three dimensions simultaneously by simply increasing its radius, as discussed above. Thus, the sedimentators of the present invention are more compact, contain much more inclined surfaces for sedimentation in a smaller footprint, and are more easily scalable cell retention devices with proven applications in mammalian cell cultures. they secrete glycoproteins, such as monoclonal antibodies, and other therapeutic proteins. The clarified harvest outlet from the upper port containing the secreted protein is collected continuously from the cell retention device, while the concentrated cells from the lower outlets are recycled back to the bioreactor, resulting in a high density perfusion bioreactor. of cells, which can be operated indefinitely (for several months of continuous perfusion operation). The continuous high titer harvest from a single 1,000-liter high-density perfusion bioreactor may be greater than the cumulative production of a large powered bioreactor (> 20,000 liters) on an annual basis .

[0212] Recombinant Chinese hamster ovary cells, which are generally used for overexpression and secretion of therapeutic glycoproteins, are cultured in a controlled 1 liter bioreactor coupled with a compact 4 "cell pelletizer as shown schematically in Figure 24. The densities of viable cells in the bioreactor, in the upper effluent of the sedimenter, and in the return from the bottom of the sedimenter to the bioreactor were measured, as soon as the perfusion operation begins in 60 hours, very few living cells they are removed from the effluent above the sedimentator and the increasing amounts of viable cells are returned to the bioreactor from the bottom of the sedimenter, consequently the viable cell density of the bioreactor (VCD) is gradually increased after the perfusion operation begins and more drastically the percentage of viability (diamonds) in the bioreactor increases when the perfusion begins.

[0213] Cell size distributions were measured on samples from the bioreactor and the upper effluent from the pelletizer on day 5 and a histogram of cell sizes measured by a Beckman-Coulter Multisize Analyzer for the bioreactor sample shows a wide distribution of cells live and possibly duplicates in sizes ranging from about 10 microns to about 30 microns with a peak of about 16 microns, an acute peak of dead cells in sizes between 8 and 9 microns and a huge tail of cells in the smaller size range less than 8 microns. Another histogram of cell / particle sizes measured by the same instrument in the sample of the effluent from the upper port of the compact cell sedimenter 208, showed an enhanced peak of dead cells in a size between 8 and 9 microns, a tail of waste from cells in sizes smaller than 8 microns and dramatically a total absence of any peak for living cells of about 16 microns. These size measurements strongly demonstrate that the upper sewage effluent

dimentor selectively removes the smaller dead cells and cell residues from the perfusion bioreactor 218, while the larger living cells are continually returned to the perfusion bioreactor (218. This selective removal of smaller dead cells and cell residues were demonstrated (Batt et al. 1990 and Searles et al. 1994) with inclined plate sedimenters. The present invention of compact cell sedimentators has again reproduced these successive results in a more compact and easier design None of the other cell retention devices currently available for mammalian cells exhibit such selectivity in removing only the smallest dead cells and cell debris Example 5 Vaccines, viruses or virus-like particles or virus production gene therapy vector

[0214] The production of vaccines, such as viruses or virus-like particles (VLPs), or gene therapy vectors, such as adeno-associated viruses (AAV), lentiviruses, etc., is usually carried out by infection and lysis of live mammalian or insect cells in a batch or fed batch bioreactor culture. Viruses or virus-like particles are released from the infected cell in a lytic process after a large intracellular production of these viruses or virus-like particles. With the large difference in the size (sub-micron or nanometer scale) of these particles compared to the size (about 5 to 20 microns) of live mammalian and insect cells, the separation of viruses or virus-like particles from the culture The batch or fed batch bioreactor is very simple. With the control of the continuous rate of collection or exit of the clarified cell culture broth that mainly contains viruses or VLPs, together with cell residues, it is also possible to retain a

smaller number of infectious particles within the bioreactor together with growing living cells to infect and continuously produce vaccines in a continuous perfusion bioreactor coupled to a sedimentation device of the present invention for the continuous collection of viruses and VLPs. Example 6 Separation and recycling of solid catalyst particles

[0215] The separation of a solid catalyst particle for recycling in the reactor and reuse in other chemical reactions of liquid catalysis phase, such as Fischer-Tropsch synthesis, has been demonstrated before with lamellar sedimenters (US Patent no. .

6,720,358, 2001). Many of these two-phase chemical reactions, involving solid catalyst particles in liquid or gaseous phase reactions, can be enhanced by the particle sedimentation devices of the present invention, which features a more compact particle separation device to accomplish the same separation and recycling of solids that has been demonstrated with lamellar sedimenters. Example 7 Harvesting of plant and algae cells

[0216] Cell cultures of recombinant plants that secrete valuable products, although not yet commercially viable, however, constitute another field of potential applications for the sedimentation devices of the present invention. Inclined sedimenters have been used in various plant cell culture applications. Such devices can be replaced by the more compact tapered spiral sedimentation devices of the present invention. With the size of plant cells larger than those of yeast or mammalian cells, the efficiency of cell separation will be greater with single plant cells or plant tissue cultures.

[0217] A more immediate commercial application of the sedimentation devices of the present invention may be in the harvesting of algae cells from large-scale cultivation ponds to harvest bi-diesel products from the interior of algal cells. The relatively diluted (acre-sized) algal cell mass in large shallow lagoons that convert solar energy into intracellular fat or fatty acid storage can be harvested easily through the tapered spiral sedimentation device of the present invention, and the concentrated algae cells can be harvested from the bottom outlet. Example 8 Municipal wastewater treatment

[0218] Large-scale municipal wastewater treatment plants (when using activated sludge or pools of multiple bacterial species for the degradation of biological and organic waste in sewage or wastewater) generally use large water tanks. sedimentation and more modern versions of these plants use lamellar sedimenters to remove clarified water from the mud. The tapered spiral sedimentation devices of the present invention can be expanded to the larger sizes required in these plants, while remaining smaller in size than the large sedimentation tanks or lamellar sedimenters used today in these treatment plants. Example 9 Lightening of industrial process water

[0219] Large-scale water treatment plants, which clean both industrial wastewater and natural sources of turbid water containing suspended solids, use large-scale sedimentation tanks or lamellar inclined sedimenters. These large-scale devices can now be replaced by devices

more compact conical spiral sedimentation positives of the present invention to achieve the same objective of clearing water for industrial reuse or municipal drinking water supply. Example 10 Capture and purification of monoclonal antibodies in granules coated with À protein

[0220] Cell culture supernatants containing monoclonal antibodies can be contacted with microspheres or granules coated with protein A (40 to 200 microns) within the present sedimenter through two different entrances, for example, granules that they enter from an upper entrance and the cell culture overflow that enters through the lower door to maximize their efficiency in contact and capture. The capture of monoclonal antibodies in protein A granules is very fast, typically under a 10-minute residence time within competitive affinity chromatography columns. The microspherical granules coated with protein A will settle quickly and can be kept in suspension and mixed well to contact the cell culture supernatant by pumping it from the bottom entrance. Depleted cell culture supernatants can be continuously removed from the top outlet of the cell sedimenters of the present invention in a batch loading operation. All granules entrained with the liquid flowing upwards will settle on the sloping surfaces and return to the lower agitated region. After loading comes close to the maximum binding capacity of the addition granules, the granules can be washed with the typical washing solution at a volume of about 3 to 5 times from the pelletizer to remove unbound host cell protein along with the dead cell residues that are present in the supernatant through the upper outlet.

[0221] After the complete washing has finished, the elution media will be pumped slowly to remove the bound antibodies in the liquid medium and the concentrated antibody solution is removed through the upper port, while the granules are retained inside the sedimenter. After elution is complete, the granules are equilibrated by pumping the equilibration solution from the bottom inlet, while the granules are held in suspension by this inlet solution. After equilibration, the next batch of cell culture supernatant is loaded into the sedimenter to repeat the above four-step process, similar to the sequence used in a chromatography column. Some advantages of using the cell sedimentation devices of the present invention to capture monoclonal antibodies are that: (i) the cell culture supernatant can be directly charged to come into contact with protein A granules, without the need to remove dead cells or cell debris generally present in the supernatant; and (ii) the most efficient immediate contact of all the granules suspended in the incoming supernatant, instead of the gradual or delayed exposure of monoclonal antibodies to the fixed bed of the granules in the posterior parts of the column. The elimination of unit operations currently required by centrifugation and / or in-depth filtration to remove dead cells and cell debris will result in significant cost savings when affinity column chromatography is replaced by capture. of affinity of antibodies for protein A granules suspended within the sedimentation devices of the modalities of the present invention.

[0222] This affinity capture of the antibody product secreted by the granules coated with protein A, followed by the washing, elution and regeneration steps, can be carried out in a sequence of batch operations in a single pelletizer or continuum.

in a sequence of sedimenters. In operation, protein A granules will flow from one sedimentation to the next sedimentation in a truly countercurrent or cross-flow operation with different cell culture broth or buffers in each sedimentation of the modalities of the present invention. Example 11 Decanter / cell sedimenter for in situ extraction of secreted organic products from cells

[0223] The production and secretion of various fragrance and flavor compounds are being metabolised in microbial yeast cells, such as Saccharomyces cerevisiae. Some of these compounds can be more toxic to cells and can be immediately extracted in an organic liquid to reduce cell toxicity, as well as increase the productivity of yeast cells. Emulsions of organic liquid containing the secreted product and the aqueous layer containing the secreted microbial cells from the agitated tank bioreactor can be pumped into the entry port of a compact cell sedimentation device of the present invention. Within the quiet areas of the sedimentator, the emulsion is easily separated into the organic layer that floats on top and is collected through the upper door and in the aqueous layer that contains the living and productive cells that sediment at the bottom and are recycled to the bioreactor through the bottom door. All cellular waste will be fractionated in the organic layer and easily removed from the top of the sedimenter. The live and productive cells in the aqueous layers are returned to the bioreactor to increase cell densities and productivity within the perfusion bioreactor. Example 12 In vitro expansion of various mammalian cells, in a compact cell sedimenter used as a perimeter bioreactor

autonomous fusion

[0224] Currently, the field of in vitro expansion of various mammalian cells such as stem cells and CAR-T cells is expanding rapidly with sterile single-use disposable culture bags as bioreactors placed on the oscillation platform for mixing or inside a CO incubator, for pH control. Such bag bioreactors are increasingly operated in the continuous perfusion mode to remove accumulated residual metabolic by-products, such as ammonia and lactate, when using microfiltration membranes as cell-holding devices in the bag to maintain high viability cell during expansion. However, during the prolonged perfusion operation, dead cells and cell debris accumulate in these bags and cannot be removed through the microfiltration membranes in the bag. The cell sedimentation devices of the present invention can be operated effectively as autonomous aerial bioreactors operated in a continuous infusion to obtain fresh nutrients and to remove metabolic waste products, as well as to selectively remove all dead cells and cell waste. The lower port can be used as an inlet for the controlled mixture of multiple CO, O2 and N2 gases to maintain the desired pH and DO in the bioreactor. The air that rises through the central portion drags or loads some of the cell culture liquid, provides a smooth mixture of nutrients in the bioreactor, and exits at the top outlet, while liquid is uncoupled in the cylindrical portion of the cell. sedimentation and recycled on conical sedimentators. The return cell culture liquid can be sampled for continuous measurements of pH, OD, for computer inputs by controlling the input gas mixture and occasional sampling for cell density and viability as desired. After the desired cell expansion, the concentrated living cells are collected through the lower port when switching the gas flow to a cell collection bag. The main advantage of the present cell pelletizer / bioreactor is that it provides for easy removal of dead cells and cell debris along with toxic metabolic waste by-products, which results in a high cell density of living cells after expansion in vitro for autologous cell therapy. Example 13 Continuous separation of precipitated and concentrated therapeutic proteins

[0225] Various therapeutic proteins (eg, insulin analog glargine and monoclonal antibodies) can be precipitated by adding simple salts (eg, zinc chloride for glargine, or ammonium sulfate for antibodies) by adjusting the pH, and other solvents (for example, m-cresol or other phenolics for glargine and ethanol for antibodies). This precipitation is a low cost alternative to chromatography in the downstream purification processes for these therapeutic proteins. Currently, these precipitation steps are carried out in batch mode, followed by centrifugation or decanting to remove the supernatant from the precipitant.

[0226] When using the separation devices of the present invention, a continuous separation process can be performed. The protein-rich harvesting medium (after removing any cells by microfiltration or centrifugation or other methods) is inserted into a compact cell sedimenter of the present invention, along with other required chemicals, such as solvents, or salts in a pH modifying solution, such as NaOH or HCl. The precipitation process will occur inside the sedimenter and the protein-rich precipitate can be continuously removed at the bottom outlet, away from the protein-depleted supernatant, which is removed.

continuously from the top outlet. Example 14 Ex vivo expansion of mesenchymal / stem stromal cells (MSCs) in microcarrier granules and purification of expanded stem cells

[0227] MSCs can expand ex vivo in the presence of the appropriate growth medium and are normally grown attached to surfaces, such as tissue culture flasks, petri dishes, roll flasks, cell cubes and microcarrier granules. The growth together in microcarrier granules (size ranging from 100 microns to 500 microns) is very easily scalable, as they are suspended in shaken or shaken bioreactors, controlled for ideal growth conditions such as pH, temperature temperature, dissolved oxygen concentration and nutrient concentrations. However, the separation of expanded stem cells from microcarriers is a challenge, requiring enzymatic detachment, removal of the additional enzyme by washing quickly, and separation of stem cells from microcarrier granules. These different steps are currently experienced when using batch processing steps that are labor-consuming and susceptible to contamination. Each of these difficult steps can be carried out more easily in the bioreactors / cell sedimentation devices of the present invention which may include the sensor probes located inside the cyclone shell. In one embodiment, the sensor probes comprise fluorescent probes to measure one or more of the pH, dissolved oxygen (DO), glucose concentrations, temperature and CO levels, within the cyclone shell. More specifically, within these sedimentation devices: (i) the excess enzyme is washed very easily or removed through the upper port by feeding on fresh nutrient medium through the lower port, while the detached cells with slower sedimentation and the newly stripped microcarrier granules of rapid sedimentation are kept in circulation within the sedimenter, (ii) the bare microcarrier granules (100 to 500 microns) will settle much more quickly than stem cells (10 to 20 microns) microns) and can be removed from the lower port while the stem cells are circulated in suspension, and (iii) finally the expanded stem cells can be harvested through the lower port at the desired concentration for subsequent cell therapy applications. Example 15 Co-culture of stromal cells in microcarrier granules to secrete the growth factors necessary to support in vitro expansion or the growth of other differentiated cells, such as T-lymphocytes or cardiomyocytes

[0228] The growth and differentiation of pluripotent stem cells into activated cardiomyocytes or lymphocytes (CAR-T cells) requires that expensive growth factors be supplemented by the growth bioreactor. This cost can be reduced by co-culturing the desired cells with the projected mesenchymal stem cells (MSCs) that secrete the desired growth factors into the growth medium. Those cells that secrete the growth factor support the growth of other desired cells, such as CAR-T cells, cardiomyocytes, etc. This co-culture can be carried out within the cell bioreactor / classifier combination devices of the present invention, and the cost of producing or expanding such cells is significantly reduced. The expanded cells can be easily removed from the coculture by feeding in the fresh medium at a rate required to remove the expanded single cells or cell aggregates, while keeping the larger microcarrier granules within the cell bioreactor / sedimenter. Example 16 Fractionation or classification of any population of mixed cells, such as bone marrow, in several distinct subpopulations with desirable or undesirable characteristics

[0229] After loading any of the bioreactors / cell sedimentation devices of the present invention with some initial bolus from a population of mixed cells (such as bone marrow cells), it is possible to feed the fresh nutrient medium at slow, steadily increasing flows, in such a way that the smaller cells (eg, platelets, red blood cells, etc.) exit through the effluent stream higher than the lower flows, followed by larger cell types (lymphocytes, mononuclear cells, etc.) at increasingly higher flows, and then by the larger types of cells (such as macrophages, megakaryocytes, etc.) at greater variations. With the increase in nutrient feed and effluent flow rates higher than slowly increasing flow rates in stages, relatively pure populations of a single desired type of cell are obtained by leaving the bioreactor cell classification device in a medium. healthy growth of the cell culture so that they can be further propagated for subsequent use. Example 17 In vitro production of universal red blood cells

[0230] New genetic engineering methods are being developed for the targeted differentiation of hematopoietic stem cells into an erythroid cell line. The proeritroblast cells, the first stage perpetrated in erythropoiesis, are quite large (12 to 20 microns), up to three times larger than a normal erythrocyte. Polychromatophilic normoblasts, the subsequent stage in the erythroid lineage, are smaller (12 to 15 microns) than proeritroblast cells. The cells of orthochromatophilic normoblasts, the cells of precursors of nucleated erythroides, are even smaller (8 to 12 microns), followed by the smaller mature enucleated red blood cells. (Geiler, C., et al., International Journal of Stem Cells, 9: 53-59). Based on the size fractionation capabilities of the bioreactors / cell classifying devices of the present invention, all larger precursor cells are retained, and only the smaller mature enucleated red blood cells are removed from the upper effluent of the device, while all larger precursor cells expand continuously within the bioreactor / cell sorter device. Example 18 Large scale platelet production

[0231] The ex vivo expansion of high ploidy megakaryocytic cells under controlled bioreactor culture conditions and their sectioning into smaller platelet cells is increasingly understood at a fundamental level (Panuganti, S., et al. , Tissue Engineering Part A, 19: 998-1014). As this understanding develops further, these necessary culture parameters can be obtained and controlled within these cell bioreactors / devices for the growth and differentiation of megakaryocytic cells, while harvesting only the smaller, mature sectioned platelets through the top outlet of the sedimenter.

[0232] To provide additional background, context, and to fulfill the requirements of the written description of 35 USC $ 112, the following references are incorporated by reference in this document in its entirety: US Patent 5,624,580, US Patent Application Publication 2009/159523, US Patent Application Publication 2011/097800, US Patent Application Publication

2012/180662, U.S. Patent Application Publication 2014/011270.

[0233] The above examples of the present invention have been presented for purposes of illustration and description. These examples are not intended to limit the invention to the form described in this document. Consequently, variations and modifications commensurate with the teachings of the description of the invention, and the skill or knowledge of the relevant technique, are within the scope of the present invention. The specific modalities described in the examples provided in this document are intended to further explain the best known method for the practice of the invention and to allow other elements skilled in the art to use the invention in such or other modalities and with the various modifications required by the particular applications or uses of the present invention. The attached claims are intended to be interpreted as including alternative modalities to the extent permitted by the prior art.

Claims (23)

1. Sedimentation device, characterized by the fact that it comprises: an upper conical portion with at least one door; a cylindrical portion; a lower conical portion with at least one door; and a stack of cones located within the sedimentation device, where each cone in the cone stack includes a small opening and a large opening, with the small opening facing one of the upper conical portion and the lower conical opening, and the stack of cones generally centered around a longitudinal axis of the sedimentation device. 2. Sedimentation device according to claim 1, characterized by the fact that at least one cone in the cone stack is composed of metal, plastic and glass. 3. Sedimentation device, according to claim 1, characterized by the fact that the upper housing still comprises a first flange configured to couple a second lower housing flange. 4. Sedimentation device according to claim 1, characterized in that the surface of a cone of the first pile of cones is at an angle between about 25 degrees and about 85 degrees in relation to the longitudinal axis. 5. Sedimentation device, according to claim 1, characterized by the fact that the first and second conical portions are concave inwards towards the longitudinal axis. 6. Sedimentation device according to claim 6, characterized by the fact that a longitudinal cross-section of a cone body forms a line with an arched shape. 7. Sedimentation device, according to claim 1, characterized by the fact that the first conical portion is concave inward towards the longitudinal axis and the second conical concave portion outward away from the longitudinal axis. 8. Sedimentation device according to claim 7, characterized by the fact that it still comprises a second pile of cones located within the sedimentation device, in which each cone of the second pile of cones includes an opening small oriented away from the first conical portion and a large opening oriented towards the first conical portion. 9. Sedimentation device according to claim 8, characterized by the fact that the cones of the second pile of cones have bodies that are concave outwardly away from the longitudinal axis. 10. Sedimentation device, according to claim 1, characterized by the fact that it still comprises a sensor to measure a condition within the sedimentation device. 11. Sedimentation device according to claim 10, characterized by the fact that the sensor comprises a fluorescent probe; and wherein one of: at least a portion of the second conical portion near the sensor is transparent or translucent; and the second conical portion is transparent or translucent. 12. Sedimentation device, according to claim 10, characterized by the fact that the sensor is operable to measure at least one of the pH, dissolved oxygen (OD), glycoside, temperature and CO , dissolved (pCO;>). 13. Method of sedimentation of particles in a suspension, characterized by the fact that it comprises: (a) the introduction of a liquid particle suspension in a sedimentation device that includes: an upper housing with a first conical portion, a first cylindrical portion and at least one door; a lower housing interconnected to the upper housing and including a second conical portion, a second cylindrical portion and at least one port; and a stack of cones located within the sedimentation device, with each cone in the cone stack including a small opening oriented towards the first conical portion and provided with bodies that are concave inwards towards a longitudinal axis of the device. sedimentation; (b) collecting a clarified liquid from at least one port of the upper casing; and (c) collecting a concentrated liquid suspension from at least one port of the lower casing. 14. Method according to claim 13, characterized in that the liquid suspension comprises at least one of a recombinant cell suspension, an alcoholic fermentation, a suspension of solid catalyst particles, a municipal waste water, water industrial waste, mammalian cells, bacterial cells, yeast cells, plant cells, algae cells, plant cells, mammalian and / or murine hybridoma cells, stem cells, CAR-T cells, red cells precursor and mature blood, cardiomyocytes or other cells susceptible to binding in microcarrier granules, brewer's yeast, and eukaryotic cells. 15. Method according to claim 13, characterized by the fact that the liquid suspension comprises at least one of: (a) recombinant microbial cells selected from small minus one from Pichia pastoris, Saccharomyces cerevisiae, Kluyverromyces lactis, Aspergillus niger, Escherichia coli, Bacillus subtilis, and other microbial cells; and (b) non-cellular particles such as one or more of microcarrier granules for the growth of joined stem cells, a microspherical granule or resin coated with affinity binder, and microspherical granules activated on the surface. 16. Method, according to claim 13, characterized by the fact that the introduction of a liquid suspension comprises directing the liquid suspension from a disposable plastic bioreactor bag to the sedimentation device. 17. Method, according to claim 13, characterized by the fact that the collected clarified liquid comprises at least one among biological molecules, organic or inorganic compounds, chemical reagents and chemical reaction products. 18. Method, according to claim 13, characterized by the fact that the collected clarified liquid comprises at least one among hydrocarbons, polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates, antibodies, glycoproteins, terpenes, isoprenoids, polyprenoids, fragrance and flavor compounds, and beer. 19. Method, according to claim 13, characterized by the fact that the clarified liquid collected comprises at least one of the biodiesel, insulin or its analogs, brazein, antibodies, growth factors, colony stimulating factors, and erythropoietin (EPO). 20. Method, according to claim 13, characterized by the fact that it further comprises the introduction of a fluid to a fluid jacket associated with at least one of the upper and lower casings. 21. Method, according to claim 13, characterized by the fact that it still comprises the collection of data from a sensor positioned inside the sedimentation device, in which the sensor is operable to measure at least one among pH, glucose, temperature and pCO, '. 22. Method, according to claim 21, characterized by the fact that it still comprises the use of the data received from the sensor to adjust one or more of the pH, the temperature, the concentration of dissolved oxygen, the dissolved carbon, and concentrations of nutrients within the sedimentation device. 23. Method, according to claim 13, characterized by the fact that it also comprises the positioning of a second cone stack inside the sedimentation device, in which the cones of the second cone stack have the bodies that are concave outwards away from the longitudinal axis of the sedimentation device. MÓ 3 8: 8 ==: It is o), á ==> h IA XÁ L A EL rr 8 nm 8 <Tl É - Es | | 7 NX IS N / À | x uv / go) ”= 8 v